Desaturase genes, enzymes encoded thereby, and uses thereof

ABSTRACT

Disclosed are isolated polynucleotides encoding an omega-3 desaturase and a delta-12 desaturase, the enzymes encoded by the isolated polynucleotides, vectors containing the isolated polynucleotides, transgenic hosts that contain the isolated polynucleotides that express the enzymes encoded thereby, methods for producing the desaturase enzymes, and method of using the enzymes to make polyunsaturated fatty acids. The isolated polynucleotides are derived from a fungus,  Saprolegnia diclina  (ATCC 56851). In particular, omega-3-desaturase may be utilized, for example, in the conversion of arachidonic acid (AA) to eicosapentaenoic acid (EPA). Delta-12 desaturase may be used, for example, in the conversion of oleic acid (OA) to linoleic (LA). EPA or polyunsaturated fatty acids produced therefrom may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

FIELD OF THE INVENTION

The present invention is directed to the identification and isolation ofnovel genes that encode enzymes involved in the synthesis ofpolyunsaturated fatty acids (PUFAs). The invention is also directed tothe novel desaturase enzymes encoded by these genes and to methods ofutilizing the genes and/or the enzymes encoded by the genes genes. Inparticular, the invention is directed to genes derived from the fungusSaprolegnia diclina (ATCC 56851) that encode a novel ω3-desaturase (alsoreferred to herein as a Δ17-desaturase) and a novel Δ12-desaturase.These enzymes catalyze the introduction of a carbon-carbon double bondbetween a particular position within a fatty acid substrate. Forexample, the novel ω3-desaturase disclosed herein catalyzes theconversion of arachidonic acid (20:4n-6) to eicosapentaenoic acid(20:5n-3) (as well as other desaturation reactions involving othersubstrates). Likewise, the novel Δ12-desaturase disclosed hereincatalyzes the conversion of oleic acid (18:1n-9) to linoleic acid(18:2n-6). The PUFAs so formed may be added to pharmaceuticalcompositions, nutritional compositions, animal feeds, or other products.

BACKGROUND

Desaturases are a class of enzymes critical in the production oflong-chain polyunsaturated fatty acids. Polyunsaturated fatty acids(PUFAs) play many roles in the proper functioning of all life forms. Forexample, PUFAs are important components of the plasma membrane of acell, where they are found in the form of phospholipids. PUFAs also areprecursors to mammalian prostacyclins, eicosanoids, leukotrienes andprostaglandins. Additionally, PUFAs are necessary for the properdevelopment of the infant brain, as well as for tissue formation andrepair in mature mammals. In view of the biological significance ofPUFAs, attempts are being made to produce them in an efficient manner.

A number of enzymes, most notably desaturases and elongases, areinvolved in PUFA biosynthesis (see FIG. 1). Elongases catalyze theaddition of a 2-carbon unit to a fatty acid substrate. Thus, forexample, an elongase (generically designated “elo” in FIG. 1) catalyzesthe conversion of γ-linolenic acid (18:3n-6) to dihomo-γ-linolenic acid(20:3n-6), as well as the conversion of stearidonic acid (18:4n-3) toeicosatetraenoic acid (20:4n-3), etc.

Desaturases catalyze the introduction of unsaturations (i.e., doublebonds) between carbon atoms within the fatty acid alkyl chain of thesubstrate. Thus, for example, linoleic acid (18:2n-6) is produced fromoleic acid (18:1n-9) by the action of a Δ12-desaturase. Similarly,γ-linolenic acid (18:3n-6) is produced from linoleic acid by the actionof a Δ6-desaturase.

Throughout the present application, PUFAs will be unambiguouslyidentified using the “omega” system of nomenclature favored byphysiologists and biochemists, as opposed to the “delta” system orI.U.P.A.C. system normally favored by chemists. In the “omega” system, aPUFA is identified by a numeric designation of the number of carbons inthe chain. This is followed by a colon and then another numericdesignation of the number of unsaturations in the molecule. This is thenfollowed by the designation “n−x,” where x is the number of carbons fromthe methyl end of the molecule where the first unsaturation is located.Each subsequence unsaturation (where there is more than one double bond)is located 3 addition carbon atoms toward the carboxyl end of themolecule. Thus, the PUFAs described herein can be described as being“methylene-interrupted” PUFAs. Where some other designation is required,deviations from the “omega” system will be noted.

Where appropriate, the action of the desaturase enzymes described hereinwill also be identified using the “omega” system. Thus, an “omega-3”desaturase catalyzes the introduction of a double bond between the twocarbons at positions 3 and 4 from the methyl end of the substrate.However, in many instances, it is more convenient to indicate theactivity of a desaturase by counting from the carboxyl end of thesubstrate. Thus, as shown in FIG. 1, a Δ9-desaturase catalyzes theintroduction of a double bond between the two carbons at positions 9 and10 from the carboxyl end of the substrate. In short, where the term“omega” is used, the position on the substrate is being designatedrelative to the methyl terminus; where the term “delta” is used, theposition on the substrate is being designated relative to the carboxylterminus.

It must be noted that mammals cannot desaturate fatty acid substratesbeyond the Δ9 position (i.e., beyond 9 carbon atoms distant from thecarboxyl terminus). Thus, for example, mammals cannot convert oleic acid(18:1n-9) into linoleic acid (18:2n-6); linoleic acid contains anunsaturation at position Δ12. Likewise, α-linolenic acid(18:3n-3)(having unsaturations at Δ12 and Δ15) cannot be synthesized bymammals. However, for example, mammals can convert α-linolenic acid intostearidonic acid (18:4n-3) by the action of a Δ6-desaturase. (SeeFIG. 1. See also PCT publication WO 96/13591; The FASEB Journal,Abstracts, Part I, Abstract 3093, page A532 (Experimental Biology 98,San Francisco, Calif., Apr. 18–22, 1998); and U.S. Pat. No. 5,552,306.)

Still referring to FIG. 1, in mammals, fungi, and algae, the stearidonicacid so formed is converted into eicosatetraenoic acid (20:4n-3) by theaction of an elongase. This PUFA can then be converted toeicosapentaenoic acid (20:5n-3) by a Δ5-desaturase. Eicosapentaenoicacid can then, in turn, be converted to ω3-docosapentaenoic acid(22:5n-3) by an elongase.

Other eukaryotes, including fungi and plants, have enzymes thatdesaturate fatty acid substrates at carbon Δ12 (see PCT publication WO94/11516 and U.S. Pat. No. 5,443,974) and at carbon delta-15 (see PCTpublication WO 93/11245). The major polyunsaturated fatty acids ofanimals therefore are either derived from diet and/or from desaturationand elongation of linoleic acid or α-linolenic acid. In view of thesedifficulties, there remains a significant need to isolate genes involvedin PUFA synthesis. Ideally, these genes would originate from speciesthat naturally produce fatty acids that are not produced naturally inmammals. These genes could then be expressed in a microbial, plant, oranimal system, which would thereby be altered to produce commercialquantities of one or more PUFAs. Thus, there is a definite need fornovel Δ12- and Δ17-desaturase enzymes, the respective genes encodingthese enzymes, as well as recombinant methods of producing theseenzymes. Additionally, a need exists for oils containing levels of PUFAsbeyond those naturally present. Access to such Δ12- and Δ17-desaturaseenzymes allows for the production of large amounts of PUFAs that cannotbe synthesized de novo in mammals. These PUFAs can be used aspharmaceutical agents and/or nutritional supplements.

All patents, patent publications and priority documents cited herein arehereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

One embodiment of the present invention encompasses an isolatednucleotide acid sequence or fragment thereof comprising or complementaryto a nucleotide sequence encoding a polypeptide having desaturaseactivity, wherein the amino acid sequence of the polypeptide has atleast 50% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO:26 and SEQ ID NO:42.

The present invention also includes an isolated nucleotide sequence (orfragment thereof) comprising or complementary to at least 50% of thenucleotide sequence selected from the group consisting of SEQ ID NO:25and SEQ ID NO:41. In particular, the sequence may be selected from thegroup consisting of SEQ ID NO:25 and SEQ ID NO:41. The sequence mayencode a functionally active desaturase which utilizes a polyunsaturatedfatty acid as a substrate. Furthermore, the nucleotide sequence may beisolated from a fungus, such as Saprolegnia diclina.

An additional embodiment of the present invention includes a purifiedpolypeptide encoded by the nucleotide sequences described above.

The present invention also includes a purified polypeptide thatdesaturates a polyunsaturated fatty acid substrate at an omega-3 carbonof the substrate and has at least 50% amino acid identity to an aminoacid sequence comprising SEQ ID NO: 26. The Polypeptide may desaturate afatty acid substrate having 20 carbon atoms.

Additionally, the present invention encompasses a purified polypeptidethat desaturates a polyunsaturated fatty acid substrate at a delta-12carbon of the substrate and has at least 50% amino acid identity to SEQID NO: 42. The polypeptide may desaturate a fatty acid substrate having18 carbon atoms.

Another embodiment of the present invention includes a method ofproducing a desaturase comprising the steps of: isolating a nucleotidesequence comprising or complementary to at least 50% of the nucleotidesequence selected from the group consisting of SEQ ID NO: 25 and SEQ IDNO: 41; constructing a vector comprising the isolated nucleotidesequence; and introducing the vector into a host cell for a time andunder conditions sufficient for expression of a desaturase encoded bythe isolated nucleotide sequence.

A further embodiment of the present invention includes a vectorcomprising: 1) an isolated nucleotide sequence corresponding to orcomplementary to at least about 50% of the nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 25 and SEQ ID NO: 41, operablylinked to b) a regulatory sequence.

Additionally, another embodiment of the present invention includes ahost cell comprising the above vector. The host cell may be, forexample, a eukaryotic cell selected from the group consisting of amammalian cell, an insect cell, a plant cell and a fungal cell. Withrespect to the host cell, expression of the isolated nucleotide sequenceof the vector may result in the host cell producing a polyunsaturatedfatty acid that is not produced in a wild-type of the host cell.

Also, the present invention encompasses a plant cell, plant, or planttissue comprising the vector described above, wherein expression of thenucleotide sequence of the vector results in production of apolyunsaturated fatty acid by the plant cell, plant or plant tissue. Thevector in the plant cell, plant or plant tissue may induce theproduction of a polyunsaturated fatty acid selected from the groupconsisting of, for example, linoleic acid, eicosatetraenoic acid andeicosapentaenoic acid. Also, the invention includes one or more plantoils or acids expressed by the plant cell, plant or plant tissue.

The invention also includes a transgenic plant comprising theabove-described vector, wherein expression of the nucleotide sequence ofthe vector results in production of a polyunsaturated fatty acid inseeds of the transgenic plant.

Another embodiment of the present invention encompasses a method forproducing a polyunsaturated fatty acid comprising the steps of:isolating a nucleotide sequence comprising or complementary to at leastabout 50% of the nucleotide sequence selected from the group consistingof SEQ ID NO: 25 and SEQ ID NO: 41; constructing a vector comprising theisolated nucleotide sequence; transforming the vector into a host cellfor a time and under conditions sufficient for expression of adesaturase encoded by the isolated nucleotide sequence; and exposing theexpressed desaturase selected from the group consisting of anomega-3-desaturase and a delta 12-desaturase, to a fatty acid substrate,whereby the substrate is catalytically converted by said desaturase intoa desired polyunsaturated fatty acid product. The substrate isdihomo-gamma-linolenic acid or arachidonic acid and the productpolyunsaturated fatty acid is eicosatetraenoic acid or eicosapentaenoicacid, respectively, when the expressed desaturase is anomega-3-desaturase. The substrate polyunsaturated fatty acid is oleicacid and the product polyunsaturated fatty acid is linoleic acid, whenthe expressed desaturase is a delta 12-desaturase.

The method may further comprise the step of exposing the polyunsaturatedfatty acid product to one or more enzymes selected from the groupconsisting of a desaturase and an elongase, whereby the polyunsaturatedfatty acid product is catalytically converted into anotherpolyunsaturated fatty acid product. The product polyunsaturated fattyacid is eicosatetraenoic acid or eicosapentaenoic acid and the anotherpolyunsaturated fatty acid is eicosapentaenoic acid or omega3-docosapentaenoic acid, respectively, when the expressed desaturase isan omega 3-desaturase. The product polyunsaturated fatty acid islinoleic acid and the another polyunsaturated fatty acid isgamma-linolenic acid, when the expressed desaturase is a delta12-desaturase.

Additionally, the method described directly above may further comprisethe step of exposing the another polyunsaturated fatty acid to one ormore enzymes selected from the group consisting of a desaturase and anelongase in order to convert the another polyunsaturated fatty acid to afinal polyunsaturated fatty acid. The final polyunsaturated fatty acidis selected from the group consisting of omega 3-docosapentaenoic acidand docosahexaenoic acid, when the expressed desaturase of step (d) isan omega 3-desaturase. In contrast, the final polyunsaturated fatty acidis selected from the group consisting of dihomo-gamma-linolenic acid,arachidonic acid, adrenic acid, omega 6-docosapentaenoic acid,eicosatetraenoic acid, stearidonic acid, eicosapentaenoic acid, omega3-docosapentaenoic acid and docosahexaenoic acid, when the expresseddesaturase is a delta 12-desaturase.

An additional embodiment of the present invention includes a method ofproducing a polyunsaturated fatty acid comprising exposing a fatty acidsubstrate to a polypeptide having at least 50% amino acid identity to anamino acid sequence selected from the group consisting of SEQ ID NO: 26and SEQ ID NO: 42, whereby the fatty acid substrate is catalyticallyconverted into the polyunsaturated fatty acid product. The fatty acidsubstrate is dihomo-gamma-linolenic acid or arachidonic acid and theproduct polyunsaturated fatty acid is eicosatetraenoic acid oreicosapentaenoic acid, respectively, when the polypeptide is an omega3-desaturase. In contrast, the fatty acid substrate is oleic acid andthe polyunsaturated fatty acid product is linoleic acid, when thepolypeptide is a delta 12-desaturase.

A further embodiment of the present invention includes a compositioncomprising at least one polyunsaturated fatty acid selected from thegroup consisting of the “product” polyunsaturated fatty acid producedaccording to the method described above, the “another” polyunsaturatedfatty acid produced according to the method described above, and the“final” polyunsaturated fatty acid produced according to the methoddescribed above. The product polyunsaturated fatty acid iseicosatetraenoic acid or eicosapentaenoic acid, when the expresseddesaturase of is an omega 3-desaturase. In contrast, the productpolyunsaturated fatty acid is linoleic acid, when the expresseddesaturase is a delta 12-desaturase. The another polyunsaturated fattyacid is eicosapentaenoic acid or omega 3-docosapentaenoic acid,respectively, when the expressed desaturase is an omega 3-desaturase.However, the another polyunsaturated fatty acid is gamma-linolenic acid,when the expressed desaturase is a delta 12-desaturase. The finalpolyunsaturated fatty acid is selected from the group consisting ofomega 3-docosapentaenoic acid and docosahexaenoic acid, when theexpressed desaturase is an omega 3-desaturase. In contrast, the finalpolyunsaturated fatty acid is selected from the group consisting ofdihomo-gamma-linolenic acid, arachidonic acid, adrenic acid, omega6-docosapentaenoic acid, eicosatetraenoic acid, stearidonic acid,eicosapentaenoic acid, omega 3-docosapentaenoic acid and docosahexaenoicacid, when the expressed desaturase is a delta 12-desaturase.

A further embodiment of the present invention includes a method ofpreventing or treating a condition caused by insufficient intake of atleast one polyunsaturated fatty acid comprising administering to thepatient the above-described composition in an amount sufficient toeffect the prevention or treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the biosynthetic pathway leading to theproduction of various PUFAs.

FIG. 2 is the nucleotide sequence of sdd17 (SEQ ID NO: 25), a genederived from S. diclina (ATCC 56851) that encodes a novel ω3-fatty aciddesaturase.

FIG. 3 is the amino acid sequence of the ω3-desaturase (SDD17) (SEQ IDNO: 26) encoded by the nucleotide sequence depicted in FIG. 2.

FIG. 4 is an amino acid sequence comparison between the SDD17 desaturasedepicted in FIG. 3 and a known Δ15-desaturase from Synechocystis sp.(SYCDESB) (SEQ ID NO: 44).

FIG. 5 is an amino acid sequence comparison between the SDD17 desaturase(SEQ ID NO: 45) depicted in FIG. 3 and a known Δ17-desaturase from C.elegans (CELEFAT) (SEQ ID NO: 46).

FIG. 6 is the nucleotide sequence of sdd12 (SEQ ID NO:41), a genederived from S. diclina (ATCC 56851) that encodes a novel Δ12-fatty aciddesaturase.

FIG. 7 is the amino acid sequence of the Δ12-desaturase (SDD12) (SEQ IDNO: 42) encoded by the nucleotide sequence depicted in FIG. 6.

FIG. 8 is an amino acid sequence comparison between the SDD12 desaturase(SEQ ID NO: 47) depicted in FIG. 7 and a known Δ12-desaturase from G.hirsutum (GHO6DES) (SEQ ID NO: 48).

FIG. 9 lists the sequence identifiers used throughout the application aswell as the corresponding amino acid or nucleotide sequence.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations Utilized Herein:

18:1n-9=oleic acid=OA

18:2n-6=linoleic acid=LA

18:3n-6=gamma-linolenic acid=GLA

18:3n-3=alpha-linolenic acid=ALA

18:4n-3=stearidonic acid=STA

20:3n-6=dihomo-gamma-linolenic acid=DGLA

20:4n-6=arachidonic acid=AA

20:4n-3=eicosatetraenoic acid=ETA

20:5n-3=eicosapentaenoic acid=EPA

22:4n-6=adrenic acid

22:5n-3=omega-3-docosapentaenoic acid=DPA

22:6n-3=docosahexaenoic acid=DHA

PUFA=polyunsaturated fatty acid

The subject invention relates to the nucleotide and translated aminoacid sequences of the ω3-desaturase and Δ12-desaturase genes isolatedfrom the fungus Saprolegnia diclina or S. diclina (ATCC 56851).Furthermore, the subject invention also includes uses of these genes andof the enzymes encoded by these genes. For example, the genes and theircorresponding enzymes may be used in the production of polyunsaturatedfatty acids such as linoleic acid, eicosapentaenoic acid, and the like.These fatty acids can be added to pharmaceutical compositions,nutritional compositions, and to other valuable products.

The fungus S. diclina (ATCC 56851), from which the polynucleotidesdescribed herein were isolated, is available commercially from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110. The fungus is supplied frozen and can be propagated in ATCCmedium 307 cornmeal agar (Difco # 0386) at 24° C. For furtherinformation on this fungus, see Beakes G. (1983) “A comparative accountof cyst coat ontogeny in saprophytic and fish-lesion (pathogenic)isolates of the Saprolegnia diclina-parasitica complex.” Can. J. Bot.61, 603–625; and Willoughby L. G., et al. (1983) “Zoospore germinationof Saprolegnia pathogenic to fish.” Trans. Br. Mycol. Soc. 80, 421–435.

The ω3-Desaturase Gene, the Δ12-Desaturase Gene, and the Enzymes EncodedThereby

The enzymes encoded by the omega-3 desaturase and delta-12 desaturasegenes of the present invention are essential in the production of PUFAshaving at least two unsaturations and an overall length of 18 carbons orlonger. The nucleotide sequence of the isolated Saprolegnia diclinaomega-3 desaturase gene is shown in SEQ ID NO: 25 and in FIG. 2. Thisgene differs significantly in sequence from all known desaturase genes,from any source. The encoded omega-3 desaturase enzyme is shown in SEQID NO: 26 and in FIG. 3. The nucleotide sequence of the isolatedSaprolegnia diclina delta-12 desaturase gene is shown in SEQ ID NO: 41and in FIG. 6. This gene also differs significantly in sequence from allknown desaturase genes, from any source. The encoded delta-12 desaturaseenzyme is shown in SEQ ID NO: 42 and in FIG. 7.

The isolated omega-3 desaturase gene of the present invention, whentransformed into a yeast host, produces an omega-3 desaturase enzymethat readily catalyzes the conversion of DGLA to ETA, AA to EPA, andadrenic acid to DPA (see Example 5). In like manner, the isolateddelta-12 desaturase gene of the present invention, when transformed intoa yeast host, produces a delta-12 desaturase enzyme that readilycatalyzes the conversion OA to LA (see Example 9).

It should be noted that the present invention also encompassesnucleotide sequences (and the corresponding encoded proteins) havingsequences comprising, identical to, or complementary to at least about50%, preferably at least about 60%, and more preferably at least about70%, even more preferably at least about 80%, and most preferably atleast about 90% of the nucleotides (i.e., having sequence identity tothe sequence) shown in SEQ ID NO: 25 and SEQ ID NO: 41 (i.e., thenucleotide sequences of the omega-3 desaturase gene and the delta-12desaturase gene of Saprolegnia diclina, respectively) described herein.(All integers between 50% and 100% are also considered to be within thescope of the present invention with respect to percent identity.) Suchsequences may be derived from any source, either isolated from a naturalsource, or produced via a semi-synthetic route, or synthesized de novo.Such sequences may be isolated from or derived from fungal sources, aswell as other non-fungal sources, such as bacterial, algal, C. elegans,mouse or human.

The present invention also encompasses fragments and derivatives of thenucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 41, as wellas fragments and derivatives of the sequences derived from othersources, and having the above-described complementarity, identity orcorrespondence. Functional equivalents of the above-sequences (i.e.,sequences having omega-3 desaturase activity or delta-12 desaturaseactivity, as appropriate) are also encompassed by the present invention.

For purposes of the present invention, a “fragment” of a nucleotidesequence is defined as a contiguous sequence of approximately at least6, preferably at least about 8, more preferably at least about 10nucleotides, and even more preferably at least about 15 nucleotidescorresponding to a region of the specified nucleotide sequence.

Sequence identity or percent identity is the number of exact matchesbetween two aligned sequences divided by the length of the shortersequence and multiplied by 100. An approximate alignment for nucleicacid sequences is provided by the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482–489 (1981). Thisalgorithm may be extended to use with peptide or protein sequences usingthe scoring matrix created by Dayhoff, Atlas of Protein Sequences andStructure, M. O. Dayhoff ed., 5 Suppl. 3:353–358, National BiomedicalResearch Foundation, Washington, D.C., USA, and normalized by Gribskov,Nucl. Acids Res. 14(6):6745–66763 (1986). The Genetics Computer Group(GCG) (Madison, Wis.) provides a computer program that automates thisalgorithm for both nucleic acid and peptide sequences in the “BestFit”utility application. The default parameters for this method aredescribed in the Wisconsin Sequence Analysis Package Program Manual,Version 8 (1995) (available from GCG). Other equally suitable programsfor calculating the percent identity or similarity between sequences aregenerally known in the art.

The invention also includes a purified polypeptide which desaturatesPUFAs at the omega-3 position and has at least about 50% amino acidsimilarity or identity, preferably at least about 60% similarity oridentity, more preferably at least about 70% similarity or identity,even more preferably at least about 80% similarity or identity, and mostpreferably at least about 90% similarity or identity to the amino acidsequence shown in SEQ ID NO: 26 (FIG. 3) and encoded by the above-notednucleotide sequence(s) (All integers between 50% and 100% similarity oridentity are also included within the scope of the invention.) Theinvention further includes a purified polypeptide which desaturatesPUFAs at the delta-12 position and has at least about 50% amino acidsimilarity or identity, preferably at least about 60% similarity oridentity, more preferably at least about 70% similarity or identity,even more preferably at least about 80% similarity or identity, and mostpreferably at least about 90% similarity or identity to the amino acidsequence shown in SEQ. ID. NO: 42 (FIG. 7) which, in turn, is encoded bythe above-described nucleotide sequence(s). (All integers between 50%and 100% similarity or identity are also included within the scope ofthe invention.)

The term “identity” refers to the relatedness of two sequences on anucleotide-by-nucleotide basis over a particular comparison window orsegment. Thus, identity is defined as the degree of sameness,correspondence or equivalence between the same strands (either sense orantisense) of two DNA segments. “Percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over aparticular region, determining the number of positions at which theidentical base occurs in both sequence in order to yield the number ofmatched positions, dividing the number of such positions by the totalnumber of positions in the segment being compared and multiplying theresult by 100. Optimal alignment of sequences may be conducted by thealgorithm of Smith & Waterman, Appl. Math. 2:482 (1981), by thealgorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by themethod of Pearson & Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988)and by computer programs which implement the relevant algorithms (e.g.,Clustal Macaw Pileup; Higgins et al., CABIOS. 5L151–153 (1989)), FASTDB(Intelligenetics), BLAST (National Center for Biomedical Information;Altschul et al., Nucleic Acids Research 25:3389–3402 (1997)), PILEUP(Genetics Computer Group, Madison, Wis.) or GAP, BESTFIT, FASTA andTFASTA (Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, Madison, Wis.). (See U.S. Pat. No. 5,912,120.)

For purposes of the present invention, “complementarity is defined asthe degree of relatedness between two DNA segments. It is determined bymeasuring the ability of the sense strand of one DNA segment tohybridize with the antisense strand of the other DNA segment, underappropriate conditions, to form a double helix. A “complement” isdefined as a sequence which pairs to a given sequence based upon thecanonic base-pairing rules. For example, a sequence A-G-T in onenucleotide strand is “complementary” to T-C-A in the other strand.

In the double helix, adenine appears in one strand, thymine appears inthe other strand. Similarly, wherever guanine is found in one strand,cytosine is found in the other. The greater the relatedness between thenucleotide sequences of two DNA segments, the greater the ability toform hybrid duplexes between the strands of the two DNA segments.

“Similarity” between two amino acid sequences is defined as the presenceof a series of identical as well as conserved amino acid residues inboth sequences. The higher the degree of similarity between two aminoacid sequences, the higher the correspondence, sameness or equivalenceof the two sequences. (“Identity between two amino acid sequences isdefined as the presence of a series of exactly alike or invariant aminoacid residues in both sequences.) The definitions of “complementarity”,“identity” and “similarity” are well known to those of ordinary skill inthe art.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 amino acids, morepreferably at least 8 amino acids, and even more preferably at least 15amino acids from a polypeptide encoded by the nucleic acid sequence.

The present invention also encompasses an isolated nucleotide sequencewhich encodes PUFA desaturase activity and that is hybridizable, undermoderately stringent conditions, to a nucleic acid having a nucleotidesequence comprising or complementary to the nucleotide sequencecomprising SEQ ID NO:25 or SEQ ID NO:41. A nucleic acid molecule is“hybridizable” to another nucleic acid molecule when a single-strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and ionicstrength (see Sambrook et al., “Molecular Cloning: A Laboratory Manual,Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.)). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. “Hybridization”requires that two nucleic acids contain complementary sequences.However, depending on the stringency of the hybridization, mismatchesbetween bases may occur. The appropriate stringency for hybridizingnucleic acids depends on the length of the nucleic acids and the degreeof complementation. Such variables are well known in the art. Morespecifically, the greater the degree of similarity or homology betweentwo nucleotide sequences, the greater the value of Tm for hybrids ofnucleic acids having those sequences. For hybrids of greater than 100nucleotides in length, equations for calculating Tm have been derived(see Sambrook et al., supra). For hybridization with shorter nucleicacids, the position of mismatches becomes more important, and the lengthof the oligonucleotide determines its specificity (see Sambrook et al.,supra).

As used herein, an “isolated nucleic acid fragment or sequence” is apolymer of RNA or DNA that is single- or double-stranded, optionallycontaining synthetic, non-natural or altered nucleotide bases. Anisolated nucleic acid fragment in the form of a polymer of DNA may becomprised of one or more segments of cDNA, genomic DNA or synthetic DNA.(A “fragment” of a specified polynucleotide refers to a polynucleotidesequence which comprises a contiguous sequence of approximately at leastabout 6 nucleotides, preferably at least about 8 nucleotides, morepreferably at least about 10 nucleotides, and even more preferably atleast about 15 nucleotides, and most preferable at least about 25nucleotides identical or complementary to a region of the specifiednucleotide sequence.) Nucleotides (usually found in their5′-monophosphate form) are referred to by their single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

The terms “fragment or subfragment that is functionally equivalent” and“functionally equivalent fragment or subfragment” are usedinterchangeably herein. These terms refer to a portion or subsequence ofan isolated nucleic acid fragment in which the ability to alter geneexpression or produce a certain phenotype is retained whether or not thefragment or subfragment encodes an active enzyme. For example, thefragment or subfragment can be used in the design of chimeric constructsto produce the desired phenotype in a transformed plant. Chimericconstructs can be designed for use in co-suppression or antisense bylinking a nucleic acid fragment or subfragment thereof, whether or notit encodes an active enzyme, in the appropriate orientation relative toa plant promoter sequence.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.

“Native gene” refers to a gene as found in nature with its ownregulatory sequences. In contrast, “chimeric construct” refers to acombination of nucleic acid fragments that are not normally foundtogether in nature. Accordingly, a chimeric construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than thatnormally found in nature. (The term “isolated” means that the sequenceis removed from its natural environment.)

A “foreign” gene refers to a gene not normally found in the hostorganism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric constructs. A “transgene” is a genethat has been introduced into the genome by a transformation procedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoter sequences canalso be located within the transcribed portions of genes, and/ordownstream of the transcribed sequences. Promoters may be derived intheir entirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (1989) Biochemistry of Plants 15:1–82. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

An “intron” is an intervening sequence in a gene that does not encode aportion of the protein sequence. Thus, such sequences are transcribedinto RNA but are then excised and are not translated. The term is alsoused for the excised RNA sequences. An “exon” is a portion of thesequence of a gene that is transcribed and is found in the maturemessenger RNA derived from the gene, but is not necessarily a part ofthe sequence that encodes the final gene product.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., (1989) PlantCell 1:671–680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a DNA that is complementary to andsynthesized from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double-strandedform using the Klenow fragment of DNA polymerase I. “Sense” RNA refersto RNA transcript that includes the mRNA and can be translated intoprotein within a cell or in vitro. “Antisense RNA” refers to an RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene (U.S.Pat. No. 5,107,065). The complementarity of an antisense RNA may be withany part of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence.“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNAthat may not be translated but yet has an effect on cellular processes.The terms “complement” and “reverse complement” are used interchangeablyherein with respect to mRNA transcripts, and are meant to define theantisense RNA of the message.

The term “endogenous RNA” refers to any RNA which is encoded by anynucleic acid sequence present in the genome of the host prior totransformation with the recombinant construct of the present invention,whether naturally-occurring or non-naturally occurring, i.e., introducedby recombinant means, mutagenesis, etc.

The term “non-naturally occurring” means artificial, not consistent withwhat is normally found in nature.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

The term “expression”, as used herein, refers to the production of afunctional end-product. Expression of a gene involves transcription ofthe gene and translation of the mRNA into a precursor or mature protein.“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. The preferredmethod of cell transformation of rice, corn and other monocots is theuse of particle-accelerated or “gene gun” transformation technology(Klein et al., (1987) Nature (London) 327:70–73; U.S. Pat. No.4,945,050), or an Agrobacterium-mediated method using an appropriate Tiplasmid containing the transgene (Ishida Y. et al., 1996, NatureBiotech. 14:745–750). The term “transformation” as used herein refers toboth stable transformation and transient transformation.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a cycle.

Polymerase chain reaction (“PCR”) is a powerful technique used toamplify DNA millions of fold, by repeated replication of a template, ina short period of time. (Mullis et al, Cold Spring Harbor Symp. Quant.Biol. 51:263–273 (1986); Erlich et al, European Patent Application50,424; European Patent Application 84,796; European Patent Application258,017, European Patent Application 237,362; Mullis, European PatentApplication 201,184, Mullis et al U.S. Pat. No. 4,683,202; Erlich, U.S.Pat. No. 4,582,788; and Saiki et al, U.S. Pat. No. 4,683,194). Theprocess utilizes sets of specific in vitro synthesized oligonucleotidesto prime DNA synthesis. The design of the primers is dependent upon thesequences of DNA that are desired to be analyzed. The technique iscarried out through many cycles (usually 20–50) of melting the templateat high temperature, allowing the primers to anneal to complementarysequences within the template and then replicating the template with DNApolymerase.

The products of PCR reactions are analyzed by separation in agarose gelsfollowed by ethidium bromide staining and visualization with UVtransillumination. Alternatively, radioactive dNTPs can be added to thePCR in order to incorporate label into the products. In this case theproducts of PCR are visualized by exposure of the gel to x-ray film. Theadded advantage of radiolabeling PCR products is that the levels ofindividual amplification products can be quantitated.

The terms “recombinant construct”, “expression construct” and“recombinant expression construct” are used interchangeably herein.These terms refer to a functional unit of genetic material that can beinserted into the genome of a cell using standard methodology well knownto one skilled in the art. Such construct may be itself or may be usedin conjunction with a vector. If a vector is used then the choice ofvector is dependent upon the method that will be used to transform hostplants as is well known to those skilled in the art. For example, aplasmid vector can be used. The skilled artisan is well aware of thegenetic elements that must be present on the vector in order tosuccessfully transform, select and propagate host cells comprising anyof the isolated nucleic acid fragments of the invention. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al., (1985) EMBO J. 4:2411–2418; De Almeida et al., (1989) Mol. Gen.Genetics 218:78–86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

Expression of the Omega-3-Desaturase and the Delta 12-Desaturase Genes

Once the genes encoding the omega-3 and delta-12 desaturase enzymes havebeen isolated, they may then be introduced into either a prokaryotic oreukaryotic host cell (individually or in combination) through the use ofa vector or construct. The vector, for example, a bacteriophage, cosmidor plasmid, may comprise the nucleotide sequence encoding either or bothof the desaturase enzymes, as well as any promoter which is functionalin the host cell and is able to elicit expression of the desaturase(s)encoded by the nucleotide sequence(s). The promoter is in operableassociation with, or operably linked, to the nucleotide sequence. (Asnoted above, a regulatory sequence (e.g., a promoter) is said to be“operably linked” with a coding sequence if the regulatory sequenceaffects transcription or expression of the coding sequence. The promoter(or other type of regulatory sequence) need not be directly linked tothe coding sequence. Suitable promoters include, for example, those fromgenes encoding alcohol dehydrogenase, glyceraldehyde-3 phosphatedehydrogenase, phosphoglucoisomerase, phosphoglycerate kinase, acidphosphatase, T7, TPI, lactase, metallothionein, cytomegalovirusimmediate early, whey acidic protein, glucoamylase, and promotersactivated in the presence of galactose, for example, GAL1 and GAL10.Additionally, nucleotide sequences which encode other proteins,oligosaccharides, lipids, etc. may also be included within the vector aswell as other regulatory sequences such as a polyadenylation signal(e.g., the poly-A signal of SV-40T-antigen, ovalalbumin or bovine growthhormone), antibiotic resistance markers, auxotrophic markers, and thelike. The choice of sequences present in the construct is dependent uponthe desired expression products, the nature of the host cell, and theproposed means to separate transformed cells from non-transformed cells.

As noted above, once the vector has been constructed, it may then beintroduced into the host cell of choice by methods known to those ofordinary skill in the art including, for example, transfection,transformation and electroporation (see Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Vol. 1–3, ed. Sambrook et al., Cold Spring HarborLaboratory Press (1989)). The host cell is then cultured under suitableconditions permitting expression of the genes leading to the productionof the desired PUFA, which is then recovered and purified. (Thesubstrates which may be produced by the host cell either naturally ortransgenically, as well as the enzymes which may be encoded by DNAsequences present in the vector, which is subsequently introduced intothe host cell, are shown in FIG. 1.)

Examples of suitable prokaryotic host cells include, for example,bacteria such as Escherichia coli and Bacillus subtilis, as well ascyanobacteria such as Spirulina spp. (i.e., blue-green algae). Examplesof suitable eukaryotic host cells include, for example, mammalian cells,plant cells, yeast cells such as Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Lipomyces starkey, Candida spp. such as Yarrowia(Candida) lipolytica, Kluyveromyces spp., Pichia spp., Trichoderma spp.or Hansenula spp., or fungal cells such as filamentous fungal cells, forexample, Aspergillus, Neurospora and Penicillium. Preferably,Saccharomyces cerevisiae (baker's yeast) cells are utilized.

Expression in a host cell can be accomplished in a transient or stablefashion. Transient expression can occur from introduced constructs whichcontain expression signals functional in the host cell, but whichconstructs do not replicate and rarely integrate into the host cell, orwhere the host cell is not proliferating. Transient expression also canbe accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest, although such inducible systemsfrequently exhibit a low basal level of expression. Stable expressioncan be achieved by introducing a construct that can integrate into thehost genome or that autonomously replicates in the host cell. Stableexpression of the gene of interest can be selected through the use of aselectable marker located on or co-transfected with the expressionconstruct, followed by selection for cells expressing the marker. Whenstable expression results from integration, the site of the construct'sintegration can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination with the host locus.Where constructs are targeted to an endogenous locus, all or some of thetranscriptional and translational regulatory regions can be provided bythe endogenous locus.

A transgenic mammal may also be used in order to express the enzymes ofthe present invention, and thus ultimately produce the PUFA(s) ofinterest. More specifically, once the above-described construct iscreated, it may be inserted into the pronucleus of an embryo. The embryomay then be implanted into a recipient female. Alternatively, a nucleartransfer method can also be utilized (Schnieke et al., Science278:2130–2133 (1997)). Gestation and birth are then permitted to occur(see, e.g., U.S. Pat. Nos. 5,750,176 and 5,700,671). Milk, tissue, orother fluid samples from the offspring should then contain alteredlevels of PUFAs, as compared to the levels normally found in thenon-transgenic animal. Subsequent generations may be monitored forproduction of the altered or enhanced levels of PUFAs and thusincorporation of the gene(s) encoding the desired desaturase enzyme(s)into their genomes. The mammal utilized as the host may be selected fromthe group consisting of, for example, mice, rats, rabbits, swine(porcines), goats and sheep (ovines), horses, and bovines. However, anymammal may be used provided it has the ability to incorporate DNAencoding the enzyme of interest into its genome.

For expression of a desaturase polypeptide, functional transcriptionaland translational initiation and termination regions are operably linkedto the DNA encoding the desaturase polypeptide of interest.Transcriptional and translational initiation and termination regions arederived from a variety of nonexclusive sources, including the DNA to beexpressed, genes known or suspected to be capable of expression in thedesired system, expression vectors, chemical synthesis, or from anendogenous locus in a host cell. Expression in a plant tissue and/orplant part presents certain efficiencies, particularly where the tissueor part is one which is harvested early, such as seed, leaves, fruits,flowers, roots, etc. Expression can be targeted to that location withthe plant by utilizing specific regulatory sequence such as those ofU.S. Pat. Nos. 5,463,174; 4,943,674; 5,106,739; 5,175,095; 5,420,034;5,188,958; and 5,589,379.

Alternatively, the expressed protein can be an enzyme that produces aproduct, and that product may be incorporated, either directly or uponfurther modifications, into a fluid fraction from the host plant.Expression of a desaturase gene, or antisense desaturase transcripts,can alter the levels of specific PUFAs, or derivatives thereof, found inplant parts and/or plant tissues. The desaturase polypeptide codingregion may be expressed either by itself or with other genes, in orderto produce tissues and/or plant parts containing higher proportions ofdesired PUFAs, or in which the PUFA composition more closely resemblesthat of human breast milk (Prieto et al., PCT publication WO 95/24494).

The termination region may be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known to be satisfactory in avariety of hosts from the same and different genera and species. Thetermination region usually is selected as a matter of convenience ratherthan because of any particular property.

As noted above, a plant (e.g., Glycine max (soybean) or Brassica napus(canola)) or plant tissue may also be utilized as a host or host cell,respectively, for expression of the desaturase enzymes, which may, inturn, be utilized in the production of PUFAs. More specifically, desiredPUFAS can be expressed in seed. Methods of isolating seed oils are knownin the art. Thus, in addition to providing a source for PUFAs, seed oilcomponents may be manipulated through the expression of the desaturasegenes, as well as perhaps other desaturase genes and elongase genes, toprovide seed oils that can be added to nutritional compositions,pharmaceutical compositions, animal feeds and cosmetics. Once again, avector that comprises a DNA sequence encoding the desaturase gene ofinterest, operably linked to a promoter, is introduced into the planttissue or plant for a time and under conditions sufficient forexpression of the desaturase gene. The vector may also comprise one ormore genes that encode other enzymes, for example, delta-5-desaturase,elongase, delta-12-desaturase, delta-15-desaturase, delta-17-desaturase,and/or delta-19-desaturase enzymes. The plant tissue or plant mayproduce the relevant substrate (e.g., adrenic acid or DPA) upon whichthe enzyme acts or a vector encoding enzymes that produce suchsubstrates may be introduced into the plant tissue, plant cell or plant.In addition, suitable substrates may be sprayed on plant tissuesexpressing the appropriate enzymes. Using these various techniques, onemay produce PUFAs by use of a plant cell, plant tissue, or plant. Itshould also be noted that the invention also encompasses a transgenicplant comprising the above-described vector, wherein expression of thenucleotide sequence(s) of the vector results in production of a desiredPUFA in, for example, the seeds of the transgenic plant.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, In: Methods for PlantMolecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif.,(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells, culturing those individualizedcells through the usual stages of embryonic development through therooted plantlet stage. Transgenic embryos and seeds are similarlyregenerated. The resulting transgenic rooted shoots are thereafterplanted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908); soybean (U.S.Pat. Nos. 5,569,834, 5,416,011, McCabe et. al., BiolTechnology 6:923(1988), Christou et al., Plant Physiol. 87:671–674 (1988)); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep.15:653–657 (1996), McKently et al., Plant Cell Rep. 14:699–703 (1995));papaya; and pea (Grant et al., Plant Cell Rep. 15:254–258, (1995)).

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204(1988), Gordon-Kamm et al., Plant Cell 2:603–618 (1990), Fromm et al.,BiolTechnology 8:833 (1990), Koziel et al., BiolTechnology 11: 194,(1993), Armstrong et al., Crop Science 35:550–557 (1995)); oat (Somerset al., BiolTechnology 10: 15 89 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppl. Genet.205:34, (1986); Part et al., Plant Mol. Biol. 32:1135–1148, (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133–141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant Cell Rep.7:379, (1988); Battraw and Hall, Plant Sci. 86:191–202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., BiolTechnology 10:691 (1992)), and wheat(Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature335:454–457 (1988); Marcotte et al., Plant Cell 1:523–532 (1989);McCarty et al., Cell 66:895–905 (1991); Hattori et al., Genes Dev.6:609–618 (1992); Goff et al., EMBO J. 9:2517–2522 (1990)).

Transient expression systems may be used to functionally dissect geneconstructs (see generally, Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor Press (1995)). It is understood that any ofthe nucleic acid molecules of the present invention can be introducedinto a plant cell in a permanent or transient manner in combination withother genetic elements such as vectors, promoters, enhancers etc.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,New York (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, New York (1998); Plant Molecular Biology: A LaboratoryManual, eds. Clark, Springer, New York (1997)).

In view of the above, the present invention encompasses a method ofproducing an omega-3 desaturase and/or a delta-12 desaturase enzyme, themethod comprising the steps of: 1) isolating the nucleotide sequence ofthe gene encoding the desired desaturase enzyme(s); 2) constructing avector comprising the nucleotide sequence(s); and 3) introducing thevector into a host cell for a time and under conditions sufficient forthe production of the desaturase enzyme(s).

The present invention also encompasses a method of producing PUFAs, themethod comprising exposing a suitable fatty acid substrate to the enzymesuch that the desaturase converts the fatty acid substrate to a desiredPUFA product. For example, when AA (20:4n-6) is exposed to the omega-3desaturase enzyme of the present invention, it is converted into EPA(20:5n-3). The EPA so formed may be converted into DPA (22:5n-3) by theaction of an elongase, and the DPA subsequently converted into DHA(22:6n-3) by a delta-4 desaturase.

Likewise, when OA (18:1n-9) is exposed to the delta-12 desaturase enzymeof the present invention, it is converted into LA (18:2n-6). The LA soformed may be converted into virtually all of the PUFAs shown in FIG. 1by the subsequent actions of suitable desaturases and/or elongases.

Uses of the Subject Desaturase Genes and Enzymes Encoded Thereby:

As noted above, the isolated desaturase genes and the desaturase enzymesencoded thereby have many uses. For example, the genes and thecorresponding enzymes may be used indirectly or directly, singly or incombination, in the production of PUFAs. For example, the omega-3desaturase may be used in the production of ETA, EPA, DPA, DHA, and thelike. As used in this context, the word “directly” encompasses thesituation where the enzyme is used to catalyze the conversion of a fattyacid substrate directly into the desired fatty acid product, without anyintermediate steps or pathway intermediates (e.g., the conversion of AAto EPA). The product so obtained is then utilized in a composition.“Indirectly” encompasses the situation where a desaturase according tothe present invention is used to catalyze the conversion of a fatty acidsubstrate into another fatty acid (i.e., a pathway intermediate) by thedesaturase (e.g., the conversion of AA to EPA) and then the latter fattyacid (the EPA) is converted to the desired fatty acid product by use ofanother desaturase or non-desaturase enzyme (e.g., the conversion of EPAto DPA by elongase). These PUFAs (i.e., those produced either directlyor indirectly by the activity of the subject desaturases) may be addedto, for example, nutritional compositions, pharmaceutical compositions,cosmetics, and animal feeds, all of which are encompassed by the presentinvention. Such uses are described in detail below.

Nutritional Compositions:

The present invention includes nutritional compositions. For purposes ofthe present invention, such compositions include any food or preparationfor human consumption (including for enteral and/or parenteralconsumption) which when taken into the body (a) serve to nourish orbuild up tissues or supply energy; and/or (b) maintain, restore orsupport adequate nutritional status or metabolic function.

The nutritional composition of the present invention comprises an oil,fatty acid ester, or fatty acid produced directly or indirectly by useof the desaturase genes disclosed herein. The composition may either bein a solid or liquid form. Additionally, the composition may includeedible macronutrients, vitamins, and/or minerals in amounts desired fora particular use. The amounts of these ingredients will vary dependingon whether the composition is intended for use with normal, healthyinfants, children, or adults, or for use with individuals havingspecialized needs, such as individuals suffering from metabolicdisorders and the like.

Examples of macronutrients that may be added to the compositions include(but are not limited to): edible fats, carbohydrates and proteins.Examples of such edible fats include (but are not limited to): coconutoil, borage oil, fungal oil, black current oil, soy oil, and mono- anddiglycerides. Examples of such carbohydrates include (but are notlimited to): glucose, edible lactose, and hydrolyzed search.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include (but are not limitedto) soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thenutritional compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the nutritional compositions of the presentinvention will be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by de novo synthesis.

Examples of nutritional compositions of the present invention include(but are not limited to): infant formulas, dietary supplements, dietarysubstitutes, and rehydration compositions. Nutritional compositions ofparticular interest include (but are not limited to) compositions forenteral and parenteral supplementation for infants, specialized infantformulas, supplements for the elderly, and supplements for those withgastrointestinal difficulties and/or malabsorption.

The nutritional composition of the present invention may also be addedto food even when supplementation of the diet is not required. Forexample, the composition may be added to food of any type, including(but not limited to): margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

In a preferred embodiment of the present invention, the nutritionalcomposition is an enteral nutritional product, more preferably, an adultor pediatric enteral nutritional product. This composition may beadministered to adults or children experiencing stress or havingspecialized needs due to chronic or acute disease states. Thecomposition may comprise, in addition to PUFAs produced according to thepresent invention, macronutrients, vitamins, and/or minerals, asdescribed previously. The macronutrients may be present in amountsequivalent to those present in human milk or on an energy basis, i.e.,on a per calorie basis.

The enteral formula, for example, may be sterilized and subsequentlyutilized on a ready-to-feed (RTF) basis or stored in a concentratedliquid or powder. The powder can be prepared by spray-drying the formulaprepared as indicated above, and reconstituting it by rehydrating theconcentrate. Adult and pediatric nutritional formulas are known in theart and are commercially available (e.g., Similac®, Ensure®, Jevity® andAlimentum® from Ross Products Division, Abbott Laboratories, Columbus,Ohio). An oil or fatty acid produced in accordance with the presentinvention may be added to any of these formulas.

The energy density of the nutritional compositions of the presentinvention, when in liquid form, may range from about 0.6 kcal to about 3kcal per ml. When in solid or powdered form, the nutritional supplementsmay contain from about 1.2 to more than 9 kcals per gram, preferablyabout 3 to 7 kcals per gm. In general, the osmolality of a liquidproduct should be less than 700 mOsm and, more preferably, less than 660mOsm.

The nutritional formula may include macronutrients, vitamins, andminerals, as noted above, in addition to the PUFAs produced inaccordance with the present invention. The presence of these additionalcomponents helps the individual ingest the minimum daily requirements ofthese elements. In addition to the provision of PUFAs, it may also bedesirable to add zinc, copper, folic acid, and antioxidants to thecomposition. It is believed that these substances boost a stressedimmune system and will therefore provide further benefits to theindividual receiving the composition. A pharmaceutical composition mayalso be supplemented with these elements.

In a more preferred embodiment, the nutritional composition comprises,in addition to antioxidants and at least one PUFA, a source ofcarbohydrate wherein at least 5 weight percent of the carbohydrate isindigestible oligosaccharide. In a more preferred embodiment, thenutritional composition additionally comprises protein, taurine, andcarnitine.

As noted above, the PUFAs produced in accordance with the presentinvention, or derivatives thereof, may be added to a dietary substituteor supplement, particularly an infant formula, for patients undergoingintravenous feeding or for preventing or treating malnutrition or otherconditions or disease states. As background, it should be noted thathuman breast milk has a fatty acid profile comprising from about 0.15%to about 0.36% DHA, from about 0.03% to about 0.13% EPA, from about0.30% to about 0.88% AA, from about 0.22% to about 0.67% DGLA, and fromabout 0.27% to about 1.04% GLA. Thus, fatty acids such as AA, EPA and/orDHA produced in accordance with the present invention can be used toalter, for example, the composition of infant formulas in order toreplicate more faithfully the PUFA content of human breast milk or toalter the presence of PUFAs normally found in a non-human mammal's milk.In particular, a composition for use a medicinal agent or foodsupplement, particularly a breast milk substitute or supplement, willpreferably comprise one or more of AA, DGLA and GLA. More preferably,the composition will comprise from about 0.3 to 30% AA, from about 0.2to 30% DGLA, and/or from about 0.2 to about 30% GLA.

Parenteral nutritional compositions comprising from about 2 to about 30%by weight fatty acids calculated as triglycerides are encompassed by thepresent invention. The preferred composition has about 1 to about 25% byweight of the total PUFA composition as GLA. Other vitamins,particularly fat-soluble vitamins such as vitamin A, D, E andL-carnitine, can optionally be included. When desired, a preservativesuch as alpha-tocopherol may be added in an amount of about 0.1% byweight.

In addition, the ratios of AA, DGLA and GLA can be adapted for aparticular given end use. When formulated as a breast milk supplement orsubstitute, a composition which comprises one or more of AA, DGLA andGLA will be provided in a ratio of from about 1:19:30 to about 6:1:0.2,respectively. For example, the breast milk of animals can vary in ratiosof AA:DGLA:GLA ranging from 1:19:30 to 6:1:0.2, which includesintermediate ratios which are preferably about 1:1:1, 1:2:1, 1:1:4. Whenproduced together in a host cell, adjusting the rate and percent ofconversion of a precursor substrate such as GLA and DGLA to AA can beused to control the PUFA ratios precisely. For example, a 5% to 10%conversion rate of DGLA to AA can be used to produce an AA to DGLA ratioof about 1:19, whereas a conversion rate of about 75% to 80% can be usedto produce an AA to DGLA ratio of about 6:1. Therefore, whether in acell culture system or in a host animal, regulating the timing, extentand specificity of desaturase expression, as well as the expression ofother desaturases and elongases, can be used to modulate PUFA levels andratios. The PUFAs produced in accordance with the present invention(e.g., AA, EPA, etc.) may then be combined with other PUFAs or othertypes of fatty acids in the desired concentrations and ratios.

Additionally, PUFAs produced in accordance with the present invention orhost cells transformed to contain and express the subject desaturasegenes may also be used as animal food supplements to alter an animal'stissue or milk fatty acid composition to one more desirable for human oranimal consumption.

Pharmaceutical Compositions:

The present invention also encompasses a pharmaceutical compositioncomprising one or more of the acids and/or resulting oils produced usingthe desaturase genes, in accordance with the methods described herein.Specifically, such a pharmaceutical composition may comprise one or moreof the PUFAs and/or oils, in combination with a standard, well-known,non-toxic pharmaceutically-acceptable carrier, adjuvant or vehicle suchas phosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquidor powder, injectible, or topical ointment or cream. Proper fluidity canbe maintained, for example, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. It mayalso be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Besides such inert diluents, thecomposition can also include adjuvants, such as wetting agents,emulsifying and suspending agents, sweetening agents, flavoring agentsand perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, PUFAs produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant PUFA(s). The antioxidant and PUFAcomponents should fit within the guidelines presented above.

For intravenous administration, the PUFAs produced in accordance withthe present invention or derivatives thereof may be incorporated intocommercial formulations such as Intralipids™. The typical normal adultplasma fatty acid profile comprises 6.64 to 9.46% AA, 1.45 to 3.11%DGLA, and 0.02 to 0.08% GLA. These PUFAs or their metabolic precursorscan be administered alone or in combination with other PUFAs to achievea normal fatty acid profile in a patient. Where desired, the individualcomponents of the formulations may be provided individually, or in kitform, for single or multiple use. A typical dosage of a particular fattyacid is from 0.1 mg to 20 g, taken from one to five times per day (up to100 g daily) and is preferably in the range of from about 10 mg to about1, 2, 5, or 10 g daily (taken in one or multiple doses).

Possible routes of administration of the pharmaceutical compositions ofthe present invention include, for example, enteral (e.g., oral andrectal) and parenteral. For example, a liquid preparation may beadministered orally or rectally. Additionally, a homogenous mixture canbe completely dispersed in water, admixed under sterile conditions withphysiologically acceptable diluents, preservatives, buffers orpropellants to form a spray or inhalant.

The route of administration will, of course, depend upon the desiredeffect. For example, if the composition is being utilized to treatrough, dry, or aging skin, to treat injured or burned skin, or to treatskin or hair affected by a disease or condition, it may be appliedtopically.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, overallhealth of the patient, past history of the patient, immune status of thepatient, etc.

With respect to form, the composition may be, for example, a solution, adispersion, a suspension, an emulsion or a sterile powder that is thenreconstituted.

The present invention also includes the treatment of various disordersby use of the pharmaceutical and/or nutritional compositions describedherein. In particular, the compositions of the present invention may beused to treat restenosis after angioplasty. Furthermore, symptoms ofinflammation, rheumatoid arthritis, asthma and psoriasis may also betreated with the compositions of the invention. Evidence also indicatesthat PUFAs may be involved in calcium metabolism; thus, the compositionsof the present invention may be utilized in the treatment or preventionof osteoporosis and of kidney or urinary tract stones.

Additionally, the compositions of the present invention may also be usedin the treatment of cancer. Malignant cells have been shown to havealtered fatty acid compositions. Addition of fatty acids has been shownto slow their growth, cause cell death and increase their susceptibilityto chemotherapeutic agents. Moreover, the compositions of the presentinvention may also be useful for treating cachexia associated withcancer.

The compositions of the present invention may also be used to treatdiabetes (see U.S. Pat. No. 4,826,877 and Horrobin et al., Am. J. Clin.Nutr. Vol. 57 (Suppl.) 732S–737S). Altered fatty acid metabolism andcomposition have been demonstrated in diabetic animals.

Furthermore, the compositions of the present invention, comprising PUFAsproduced either directly or indirectly through the use of the desaturaseenzymes, may also be used in the treatment of eczema and in thereduction of blood pressure. Additionally, the compositions of thepresent invention may be used to inhibit platelet aggregation, to inducevasodilation, to reduce cholesterol levels, to inhibit proliferation ofvessel wall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp.Med. Biol. Vol. 83, p. 85–101, 1976), to reduce or to preventgastrointestinal bleeding and other side effects of non-steroidalanti-inflammatory drugs (see U.S. Pat. No. 4,666,701), to prevent or totreat endometriosis and premenstrual syndrome (see U.S. Pat. No.4,758,592), and to treat myalgic encephalomyelitis and chronic fatigueafter viral infections (see U.S. Pat. No. 5,116,871).

Further uses of the compositions of the present invention include use inthe treatment of AIDS, multiple sclerosis, and inflammatory skindisorders, as well as for maintenance of general health.

Additionally, the composition of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or a PUFA produced accordingto the subject invention may be used as the sole “active” ingredient ina cosmetic composition.

Veterinary Applications:

It should be noted that the above-described pharmaceutical andnutritional compositions may be utilized in connection with animals(i.e., domestic or non-domestic, including mammals, birds, reptiles,lizards, etc.), as well as humans, as animals experience many of thesame needs and conditions as humans. For example, the oil or fatty acidsof the present invention may be utilized in animal feed supplements,animal feed substitutes, animal vitamins or in animal topical ointments.

The present invention may be further illustrated by the non-limitingexamples presented below:

EXAMPLE 1 Construction of Saprolegnia diclina (ATCC 56851) cDNA Library

To isolate genes encoding for functional desaturase enzymes, a cDNAlibrary was constructed. Saprolegnia diclina cultures were grown inpotato dextrose media (Difco # 336, BD Diagnostic Systems, Sparks, Md.)at room temperature for four days with constant agitation. The myceliawere harvested by filtration through several layers of cheesecloth, andthe cultures were crushed in liquid nitrogen using a mortar and pestle.The cell lysates were resuspended in RT buffer (Qiagen, Valencia,Calif.) containing β-mercaptoethanol and incubated at 55° C. for threeminutes. These lysates were homogenized either by repeated aspirationsthrough a syringe or over a “Qiashredder”-brand column (Qiagen). Thetotal RNA was finally purified using the “RNeasy Maxi”-brand kit(Qiagen), as per the manufacturer's protocol.

mRNA was isolated from total RNA from each organism using an oligo dTcellulose resin. The “pBluescript II XR”-brand library construction kit(Stratagene, La Jolla, Calif.) was used to synthesize double-strandedcDNA. The double-stranded cDNA was then directionally cloned (5′EcoRI/3′ XhoI) into pBluescript II SK(+) vector (Stratagene). The S.diclina library contained approximately 2.5×10⁶ clones, each with anaverage insert size of approximately 700 bp. Genomic DNA of S. diclinawas isolated by crushing the culture in liquid nitrogen followed bypurification using the “Genomic DNA Extraction”-brand kit (Qiagen), asper the manufacturer's protocol.

EXAMPLE 2 Determination of Codon Usage in Saprolegnia diclina

The 5′ ends of 350 random cDNA clones were sequenced from theSaprolegnia diclina cDNA library described in Example 1. The sequenceswere translated into six reading frames using GCG program (GeneticsComputer Group, Madison, Wis.) with the “FastA”-brand algorithm tosearch for similarity between a query sequence and a group of sequencesof the same type, specifically within the GenBank database. Many of theclones were identified as putative housekeeping genes based on proteinhomology to known genes. Eight S. diclina cDNA sequences were thusselected. Additionally, the full-length S. diclina delta 5-desaturaseand delta 6-desaturase sequences were also used (see Table 1 below).These sequences were then used to generate the S. diclina codon biastable shown in Table 2 below by employing the “CodonFrequency” programfrom GCG.

TABLE 1 Genes from Saprolegnia diclina used for generation of Codon BiasTable # amino Clone # Match # bases acids 3 Actin gene 615 205 20Ribosomal protein L23 420 140 55 Heat Shock protein 70 gene 468 156 83Glyceraldehyde-3-P-dehydrogenase 588 196 gene 138 Ribosomal protein S13gene 329 110 179 Alpha-tubulin 3 gene 591 197 190 Casein kinase II alphasubunit 627 209 gene 250 Cyclophilin gene 489 163 Delta 6-desaturase1362 453 Delta 5-desaturase 1413 471 Total 6573 2191

TABLE 2 Codon Bias Table for Saprolegnia diclina Amino acid Codon Bias %used Ala GCC 55% Arg CGC 50% Asn AAC 94% Asp GAC 85% Cys TGC 77% Gln CAG90% Glu GAG 80% Gly GGC 67% His CAC 86% Ile ATC 82% Leu CTC 52% Lys AAG87% Met ATG 100% Phe TTC 72% Pro CCG 55% Ser TCG 47% Thr ACG 46% Trp TGG100% Tyr TAC 90% Val GTC 73% Stop TGA 67%

EXAMPLE 3 Design of Degenerate Oligonucleotides for the Isolation of anOmega-3 Desaturase from Saprolegnia diclina (ATCC 56851)

Fungi like Saprolegnia diclina produce a wide range of PUFAs, includingarachidonic acid (AA) and eicosapentaenoic acid (EPA) via the PUFAbiosynthetic pathway depicted in FIG. 1. Analysis of the fatty acidcomposition of Saprolegnia diclina (ATCC 56851) showed 15.42% of thetotal lipid to be AA and 12.2% of the total lipid to be EPA (see Table5). Linoleic acid (LA) was the only other intermediate present in highamounts. This indicates that S. diclina has very active delta-6 anddelta-5 desaturases, as well as elongases that shunt intermediatesthrough the pathway depicted in FIG. 1. Due to the high percentage ofEPA in this organism, an active omega-3 desaturase (synonymous with a“delta-15” desaturase when the substrate is a C₁₈ fatty acid, a“delta-17” desaturase when the substrate is a C₂₀ fatty acid, and a“delta-19” desaturase when the substrate is a C₂₂ fatty acid) ispredicted to exist which is capable of converting AA (20:4n-6) to EPA(20:5n-3).

As just noted, omega-3 desaturases are enzymes that catalyze theintroduction of a double bond at the delta-15 position for C₁₈-acylchains, the delta-17 position for C₂₀-acyl chains, and the delta-19position for C₂₂-acyl chains. There are several known omega-3desaturases from plants, but these act exclusively on C₁₈ fatty acidsubstrates like LA (18:2n-6) and GLA (18:3n-6). These types ofdesaturases are collectively referred to as delta 15-desaturases. Atthis point in time, only one omega-3 desaturase gene has been isolatedwhose encoded enzyme catalyzes the desaturation of C₁₈, C₂₀, and C₂₂fatty acid substrates. This is fat-1 from C. elegans. See U.S. Pat. No.6,194,167, issued Feb. 27, 2001.

The approach used in this Example to identify an omega-3 desaturase fromS. diclina involved PCR amplification of a region of the desaturase geneusing degenerate oligonucleotides (primers) that contained conservedmotifs present in other known omega-3 desaturases. Omega-3 desaturasesfrom the following organisms were used for the design of thesedegenerate primers: Arabidopsis thaliana (Swissprot #P46310), Ricunuscommunis (Swissprot #P48619), Glycine max (Swissprot #P48621), Sesamumindicum (Swissprot #P48620), Nicotiana tabacum (GenBank # D79979),Perilla frutescens (GenBank # U59477), Capsicum annuum (GenBank #AF222989), Limnanthes douglassi (GenBank # U17063), and Caenorhabditiselegans (GenBank # L41807). Some primers were designed to contain theconserved histidine-box motifs that are important components of theactive site of the enzymes. See Shanklin, J. E., McDonough, V. M., andMartin, C. E. (1994) Biochemistry 33, 12787–12794.

Alignment of sequences was carried out using the CLUSTALW MultipleSequence Alignment Program (http://workbench.sdsc.edu).

The following degenerate primers were designed and used in variouscombinations:

Protein Motif 1: NH₃— TRAAIPKHCWVK —COOH Primer RO 1144 (Forward):5′-ATC CGC GCC GCC ATC CCC AAG CAC (SEQ ID NO: 1) TGC TGG GTC AAG-3′.

Protein Motif 2: NH₃— ALFVLGHDCGHGSFS —COOH (SEQ ID NO: 50)

This primer contains the histidine-box 1 (underlined).

Primer RO 1119 (Forward): 5′-GCC CTC TTC GTC CTC GGC CAY (SEQ. ID. NO:2) GAC TGC GGC CAY GGC TCG TTC TCG-3′. Primer RO 1118 (Reverse): 5′-GAGRTG GTA RTG GGG GAT CTG GGG (SEQ. ID. NO: 3) GAA GAR RTG RTG GRY GACRTG-3′.

Protein Motif 3: NH₃— PYHGWRISHRTHHQN —COOH (SEQ ID NO: 51)

This primer contains the histidine-box 2 (underlined).

Primer RO 1121 (Forward): 5′-CCC TAC CAY GGC TGG CGC ATC TCG (SEQ. ID.NO: 4) CAY CGC ACC CAY CAY CAG AAC-3′. Primer RO 1122 (Reverse): 5′-GTTCTG RTG RTG GGT CCG RTG CGA (SEQ. ID. NO: 5) GAT GCG CCA GCC RTG GTAGGG-3′. Protein Motif 4: NH₃— GSHF D/H P D/Y SDLFV —COOH Primer RO 1146(Forward): 5′-GGC TCG CAC TTC SAC CCC KAC (SEQ. ID. NO: 6) TCG GAC CTCTTC GTC-3′. Primer RO 1147 (Reverse): 5′-GAC GAA GAG GTC CGA GTM GGG(SEQ. ID. NO: 7) GTW GAA GTG CGA GCC-3′. Protein Motif 5: NH₃— WS Y/FL/V RGGLTT L/I DR —COOH Primer RO 1148 (Reverse): 5′-GCG CTG GAK GGT GGTGAG GCC (SEQ. ID. NO: 8) GCC GCG GAW GSA CGA CCA-3′.

Protein Motif 6: NH₃— HHDIGTHVIHHLFPQ —COOH (SEQ ID NO: 54)

This sequence contains the third histidine-box (underlined).

Primer RO 1114 (Reverse): 5′-CTG GGG GAA GAG RTG RTG GAT (SEQ. ID. NO:9) GAC RTG GGT GCC GAT GTC RTG RTG-3′. Protein Motif 7: NH₃— H L/F FPQ/K IPHYHL V/I EAT —COOH Primer RO 1116 (Reverse): 5′-GGT GGC CTC GAYGAG RTG GTA (SEQ. ID. NO: 10) RTG GGG GAT CTK GGG GAA GAR RTG-3′.

Protein Motif 8: NH₃— HV A/I HH L/F FPQIPHYHL —COOH (SEQ ID NO: 56)

This primer contains the third histidine-box (underlined) and accountsfor differences between the plant omege-3 desaturases and the C. elegansomega-3-desaturase.

Primer RO 1118 (Reverse): 5′-GAG RTG GTA RTG GGG GAT CTG GGG (SEQ. ID.NO: 11) GAA GAR RTG RTG GRY GAC RTG-3′.

The degeneracy code used for SEQ. ID. NOS: 1 through 11 was as follows:R=A/G; Y=C/T; M=A/C; K=G/T; W=A/T; S=C/G; B=C/G/T; D=A/G/T; H=A/C/T;V=A/C/G; and N=A/C/G/T.

EXAMPLE 4 Identification and Isolation of an Omega-3 Desaturase Genefrom Saprolegnia diclina (ATCC 56851)

Various sets of the degenerate primers disclosed in Example 3 were usedin PCR amplification reactions, using as a template either the S.diclina cDNA library plasmid DNA (from Example 1), or S. diclina genomicDNA. Also various different DNA polymerases and reaction conditions wereused for the PCR amplifications. These reactions variously involvedusing “Platinum Taq”-brand DNA polymerase (Life Technologies Inc.,Rockville, Md.), or cDNA polymerase (Clonetech, Palo Alto, Calif.), orTaq PCR-mix (Qiagen), at differing annealing temperatures.

PCR amplification using the primers RO 1121 (Forward) (SEQ. ID. NO: 4)and RO 1116 (Reverse) (SEQ. ID. NO: 10) resulted in the successfulamplification of a fragment homologous to a known omega-3 desaturase.The RO 1121 (Forward) primer corresponds to the protein motif 3; the RO1116 (Reverse) primer corresponds to the protein motif 7.

PCR amplification was carried out in a 50 μl total volume containing: 3μl of the cDNA library template, PCR buffer containing 40 mM Tricine-KOH(pH 9.2), 15 mM KOAc, 3.5 mM Mg(OAc)₂, 3.75 μg/ml BSA (finalconcentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmoleof each primer and 0.5 μl of “Advantage”-brand cDNA polymerase(Clonetech). Amplification was carried out as follows: initialdenaturation at 94° C. for 3 minutes, followed by 35 cycles of thefollowing: 94° C. for 1 min, 60° C. for 30 sec, 72° C. for 1 min. Afinal extension cycle of 72° C. for 7 min was carried out, followed byreaction termination at 4° C.

A single ˜480 bp PCR band was generated which was resolved on a 1%“SeaKem Gold”-brand agarose gel (FMC BioProducts, Rockland, Me.), andgel-purified using the Qiagen Gel Extraction Kit. The staggered ends onthe fragment were “filled-in” using T4 DNA polymerase (LifeTechnologies, Rockville, Md.) as per the manufacturer's instructions,and the DNA fragments were cloned into the PCR-Blunt vector (Invitrogen,Carlsbad, Calif.). The recombinant plasmids were transformed into TOP10supercompetent cells (Invitrogen), and eight clones were sequenced and adatabase search (Gen-EMBL) was carried out.

Clones “sdd17-7-1” to “sdd17-7-8’ were all found to contain and ˜483 bpinsert. The deduced amino acid sequence from this fragment showedhighest identity to the following proteins (based on a “tFastA” search):

1. 37.9% identity in 161 amino acid overlap with an omega-3 (delta-15)desaturase from Synechocystis sp. (Accession # D13780).

2. 40.7% identity in 113 amino acid overlap with Picea abies plastidialomega-3 desaturase (Accession # AJ302017).

3. 35.9% identity in 128 amino acid overlap with Zea mays FAD8 fattyacid desaturase (Accession # D63953).

Based on its sequence homology to known omega-3 fatty acid desaturases,it seemed likely that this DNA fragment was part of an omega-3desaturase gene present in S. diclina.

The DNA sequence identified above was used in the designoligonucleotides to isolate the 3′ and the 5′ ends of this gene from thecDNA library described in Example 1. To isolate the 3′ end of the gene,the following oligonucleotides were designed:

RO 1188 (Forward): 5′-TAC GCG TAC CTC ACG TAC TCG CTC G-3′. (SEQ. ID.NO: 12) RO 1189 (Forward): 5′-TTC TTG CAC CAC AAC GAC GAA GCG ACG-3′.(SEQ. ID. NO: 13) RO 1190 (Forward): 5′-GGA GTG GAC GTA CGT CAA GGG CAAC-3′. (SEQ. ID. NO: 14) RO 1191 (Forward): 5′-TCA AGG GCA ACC TCT CGAGCG TCG AC-3′. (SEQ. ID. NO: 15)

These primers (SEQ. ID. NOS: 12–15) were used in combinations with thepBluescript SK(+) vector oligonucleotide: RO 898: 5′-CCC AGT CAC GAC GTTGTA AAA CGA CGG CCA G-3′ (SEQ. ID. NO: 16).

PCR amplifications were carried out using either the “Taq PCR MasterMix” brand polymerase (Qiagen) or “Advantage”-brand cDNA polymerase(Clonetech) or “Platinum”-brand Taq DNA polymerase (Life Technologies),as follows:

For the “Taq PCR Master Mix” polymerase, 10 pmoles of each primer wereused along with 1 μl of the cDNA library DNA from Example 1.Amplification was carried out as follows: initial denaturation at 94° C.for 3 min, followed by 35 cycles of the following: 94° C. for 1 min, 60°C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for 7min was carried out, followed by the reaction termination at 4° C. Thisamplification resulted in the most distinct bands as compared with theother two conditions tested.

For the “Advantage”-brand cDNA polymerase reaction, PCR amplificationwas carried out in a 50 μl total volume containing: 1 μl of the cDNAlibrary template from Example 1, PCR buffer containing 40 mM Tricine-KOH(pH 9.2), 15 mM KOAc, 3.5 mM Mg(OAc)₂, 3.75 μg/ml BSA (finalconcentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmoleof each primer and 0.5 μl of cDNA polymerase (Clonetech). Amplificationwas carried out as described for the Taq PCR Master Mix.

For the “Platinum”-brand Taq DNA polymerase reaction, PCR amplificationwas carried out in a 50 μl total volume containing: 1 μl of the cDNAlibrary template from Example 1, PCR buffer containing 20 mM Tris-Cl, pH8.4, 50 mM KCl (final concentration), 200 μM each deoxyribonucleotidetriphosphate, 10 pmole of each primer, 1.5 mM MgSO₄, and 0.5 μl ofPlatinum Taq DNA polymerase. Amplification was carried out as follows:initial denaturation at 94° C. for 3 min, followed by 30 cycles of thefollowing: 94° C. for 45 sec, 55° C. for 30 sec, 68° C. for 2 min. Thereaction was terminated at 4° C.

All four sets of primers in combination with the vector primer generateddistinct bands. PCR bands from the combination (RO 1188+RO 898)were >500 bp and this was gel-purified and cloned separately. The PCRbands generated from the other primer combinations were <500 bp. Thebands were gel-purified, pooled together, and cloned into PCR-Bluntvector (Invitrogen) as described earlier. The recombinant plasmids weretransformed into TOP10 supercompetent cells (Invitrogen) and clones weresequenced and analyzed.

Clone “sdd17-16-4” and “sdd16-6” containing the ˜500 bp insert, andclones “sdd17-17-6,” “sdd17-17-10,” and “sdd17-20-3,” containing the˜400 bp inserts, were all found to contain the 3′-end of the putativeomega-3 desaturase. These sequences overlapped with each other, as wellas with the originally identified fragment of this putative omega-3desaturase gene. All of the sequences contained the ‘TAA’ stop codon anda poly-A tail typical of 3′-ends of eukaryotic genes. This 3′-endsequence was homologous to other known omega-3 desaturases, sharing thehighest identity (41.5% in 130 amino acid overlap) with theSynechocystis delta-15 desaturase (Accession # D13780).

For the isolation of the 5′-end of the this gene, the followingoligonucleotides were designed and used in combinations with thepBluescript SK(+) vector oligonucleotide:

RO 899: 5′-AGC GGA TAA CAA TTT CAC ACA GGA AAC AGC -3′. (SEQ. ID. NO:17) RO 1185 (Reverse): 5′-GGT AAA AGA TCT CGT CCT TGT CGA TGT (SEQ. ID.NO: 18) TGC-3′. RO 1186 (Reverse): 5′-GTC AAA GTG GCT CAT CGT GC-3′.(SEQ. ID. NO: 19) RO 1187 (Reverse): 5′-CGA GCG AGT ACG TGA GGT ACG CGTAC-3′. (SEQ. ID. NO: 20)

Amplifications were carried out using either the “Taq PCR MasterMix”-brand polymerase (Qiagen) or the “Advantage”-brand cDNA polymerase(Clonetech) or the “Platinum”-brand Taq DNA polymerase (LifeTechnologies), as described hereinabove for the 3′ end isolation.

PCR bands generated from primer combinations (RO 1185 or RO 1186+RO 899)were between ˜580 to ˜440 bp and these were pooled and cloned intoPCR-Blunt vector as described above. Clones thus generated included“sdd17-14-1,” “sdd17-14-10,” “sdd17-18-2,” and “sdd17-18-8,” all ofwhich showed homology with known omega-3 desaturases.

Additionally, bands generated from (RO 1187+RO 899) were ˜680 bp, andthese were cloned separately to generate clones “sdd17-22-2” and“sdd17-22-5” which also showed homology with known omega-3 desaturases.All these clones overlapped with each other, as well as with theoriginal fragment of the unknown putative omega-3 desaturase. Thesesequences contained an ATG′ site followed by an open reading frame,indicating that it is the start site of this gene. These fragmentsshowed highest identity (39.7% in 146 amino acid overlap) with thedelta-15 desaturase from Calendula officinalis (Accession # AJ245938).

The full-length of this omega-3 desaturase was obtained by PCRamplification of the S. diclina cDNA library using the followingoligonucleotides:

RO 1212 (Forward): 5′-TCA ACA GAA TTC ATG ACC GAG GAT AAG ACG AAG GTCGAG TTC CCG-3′ (SEQ. ID. NO: 21). This primer contains the ‘ATG’ startsite (single underline) followed by the 5′ sequence of the omega-3desaturase. In addition, an EcoRI site (double underline) was introducedupstream of the start site to facilitate cloning into the yeastexpression vector pYX242.

RO 1213 (Reverse): 5′-AAA AGA AAG CTT CGC TTC CTA GTC TTA GTC CGA CTTGGC CTT GGC-3′ (SEQ. ID. NO: 22). This primer contains the ‘TAA’ stopcodon (single underline) of the gene as well as sequence downstream fromthe stop codon. This sequence was included because regions within thegene were very G+C rich, and thus could not be included in the design ofoligonucleotides for PCR amplification. In addition, a HindIII site(double underline) was included for convenient cloning into a yeastexpression vector pYX242.

PCR amplification was carried out using the “Taq PCR Master Mix”-brandpolymerase (Qiagen), 10 pmoles of each primer, and 1 μl of the cDNAlibrary DNA from Example 1. Amplification was carried out as follows:initial denaturation at 94° C. for 3 min, followed by 35 cycles of thefollowing: 94° C. for 1 min, 60° C. for 30 sec, 72° C. for 1 min. Afinal extension cycle of 72° C. for 7 min was carried out, followed bythe reaction termination at 4° C.

A PCR band of ˜1 kb was thus obtained and this band was isolated,purified, cloned into PCR-Blunt vector (Invitrogen), and transformedinto TOP10 cells. The inserts were sequenced to verify the genesequence. Clone “sdd17-27–2” was digested with EcoRI/HindIII to releasethe full-length insert, and this insert was cloned into yeast expressionvector pYX242, previously digested with EcoRI/HindIII. This constructcontained 1077 bp of sdd17 cloned into pYX242. This construct waslabeled pRSP19, which was transformed into yeast SC334 for expressionstudies.

In addition, the S. diclina omega-3 gene was cloned into another yeastexpression vector, pYES2 (Invitrogen). For this, the omega-3 desaturasegene was isolated from the cDNA library generated in Example 1 by PCRamplification (as described above) using the following oligonucleotides:

RO1221 (Forward) (SEQ. ID. NO: 23) 5′-TCA ACA AAG CTT ATG ACC GAG GATAAG ACG AAG GTC GAG TTC CCG-3′ This primer contains the ‘ATG’ start site(underlined) followed by the 5′ sequence of the omega-3 desaturase. Inaddition, a HindIII site (bold) was introduced upstream of the startsite to facilitate cloning into the pYES2 yeast expression vector.

RO1222 (Reverse) (SEQ. ID. NO: 24) 5′-AAA AGA GAA TTC CGC TTC CTA GTCTTA GTC CGA CTT GGC CTT GGC-3′ This primer contains the ‘TAA’ stop codon(underlined) of the gene as well as sequence downstream from the stopcodon. This sequence was included since regions within the gene werevery G+C rich, and thus could not be included in the design ofoligonucleotides for PCR amplification. In addition, an EcoRI site(bold) was included for convenient cloning into the pYES2 yeastexpression vector.

The ˜1 kb PCR band thus generated was digested with HindIII/EcoRI, andcloned into pYES2 digested with the same restriction enzymes. Theresulting construct (sdd17+pYES2) was labeled pRSP20, and was used inco-expression studies.

Attempts were also made to isolate the full-length sdd17 gene fromgenomic DNA by PCR amplification. However, the PCR product obtained waslarger than 1077 bp (˜1.15 kb), and sequencing of this product revealedthe presence of small introns in the genomic sequence.

The full-length gene of this putative omega-3 desaturase (labeled sdd17)was 1077 bp in length and is shown in FIG. 2 (SEQ ID NO: 25).

The gene of SEQ ID NO: 25 encoded a protein of 358 amino acid residues(SEQ. ID. NO: 26) (FIG. 3). A search of the deduced protein sequence ofsdd17 (using the “tFastA” program) showed the protein to have highestidentity (41% in 269 amino acid overlap) with the delta-15 desaturasefrom Synechocystis sp. (ATCC Accession No. 13780) (FIG. 4) andSynechocystis sp. PCC6803 (ATCC Accession No. D90913). This proteinshared sequence similarities with several other plant omega-3desaturases. Comparison of this predicted protein sequence with the FAT1enzyme from C. elegans (ATCC Accession L41807) revealed only a 31.6%identity in 310 amino acid overlap (FIG. 5).

Like all omega-3 desaturases, this enzyme does not contain a cytochromeb5 domain within the 5′ end of its sequence. The cytochrome b5 domain ispresent in most “front-end” desaturating enzymes like delta 5- and delta6-desaturases. The omega-3 desaturase described in this example includesthe three histidine-rich sequences that are present in allmembrane-bound desaturases. These three domains are present at position89 to 93 (HDCGH), 125 to 129 (HRHHH), and 284 to 288 (HQVHH) of SEQ. ID.NO: 26. These histidine-rich boxes are believed to co-ordinate thediiron-oxo structure at the enzyme's active site, and are necessary forenzyme activity; see Stukey, J. E., McDonough, V. M. & Martn, C. E.(1990) J. Biol. Chem. 265, 20144–20149. These features are consistentwith the “SDD17” protein being a member of the membrane-bounddesaturase/hydroxylase family of the diiron-oxo proteins. The G+Ccontent of this gene is 61.8%.

EXAMPLE 5 Expression of the Omega-3 Desaturase Gene (“sdd17”) fromSaprolegnia diclina in Bakers' Yeast

To determine the substrate specificity and the class of reactioncatalyzed by the SDD17-protein, sdd17 was heterologously expressed in aSaccharomyces cerevisiae (SC334). Because S. cerevisiae cannotsynthesize fatty acids beyond OA (18:1n-9), it is an ideal system to useto determine enzyme activity on substrates longer than OA because nobackground enzyme activity will be detected. Suitable fatty acidsubstrates can be exogenously supplied to the host which are taken up bythe cell and acted on by the expressed protein of the transformed sdd17gene.

Clone pRSP19, which contained the full-length omega-3 desaturase (sdd17)from S. diclina cloned into pYX242, was transformed into Saccharomycescerevisiae (SC334) using the “Alkali-Cation Yeast Transformation”-brandkit (BIO 101, Vista, Calif.). Transformants were selected for leucineauxotrophy on media lacking leucine (DOB [-Leu]). To detect the specificdesaturase activity of these clones, transformants were grown in thepresence of 50 μM of each of LA (18:2n-6), GLA (18:3n-6), DGLA(20:3n-6), AA (20:4n-6), and adrenic acid (22:4n-6). Conversion of theseexogenously supplied fatty acid substrates into a product having oneadditional unsaturation indicates the presence of a specific desaturaseactivity that is not found in the wild-type S. cerevisiae:

Conversion of LA (18:2n-6) to ALA (18:3n-3) indicates delta-15desaturase activity.

Conversion of GLA (18:3n-6) to STA(18:4n-3) indicates delta-15desaturase activity.

Conversion of DGLA (20:3n-6) to ETA (20:4n-3) indicates delta-17desaturase activity.

Conversion of AA (20:4n-6) to EPA (20:5n-3) indicates delta-17desaturase activity.

Conversion of adrenic acid (22:4n-6) to DPA (22:5n-3) indicates delta-19desaturase activity.

The negative control strain was S. cerevisiae transformed with thepYX242 vector. The experimental cultures and the control cultures weregrown simultaneously and analyzed.

The cultures were vigorously agitated (250 rpm) and grown for 48 hoursat 24° C. in the presence of 50 μM (final concentration) of the varioussubstrates (see Table 3). The cells were spun down, washed once indistilled water, and the pellets resuspended in methanol; chloroform wasadded along with tritridecanoin (as an internal standard). Thesemixtures were incubated for at least an hour at room temperature, or at4° C. overnight. The chloroform layer was extracted and filtered througha Whatman filter with 1 gm anhydrous sodium sulfate to removeparticulate matter and residual water. The organic solvents wereevaporated at 40° C. under a stream of nitrogen. The extracted lipidswere then converted to fatty acid methyl esters (FAME) for gaschromatography analysis (GC) by adding 2 ml 0.5 N potassium hydroxide inmethanol to a closed tube. The samples were heated to 95° C.–100° C. for30 minutes and cooled to room temperature. Approximately 2 ml 14%borontrifluoride in methanol was added and the heating repeated. Afterthe extracted lipid mixture cooled, 2 ml of water and 1 ml of hexanewere added to extract the FAME for analysis by GC. The percentconversion was calculated using the formula:

TABLE 3 Yeast Expression of the Omega- 3 Desaturase (SDD17) fromSaprolegnia diclina at 24° C. Substrate* Product % Enzyme CloneIncorporated Produced Conversion Activity PRSP19 18:2 n-6 18:3 n-3 0Delta 15 (9.42%) (0%) 18:3 n-6 18:4 n-3 0 Delta 15 (9.11%) (0%) 20:3 n-620:4 n-3 5% Delta 17 (21.36%) (1.18%) 20:4 n-6 20:5 n-3 13.8% Delta 17(32.14%) (5.16%) 22:4 n-6 22:5 n-3 4% Delta 19 (28.65%) (1.22%) Control18:2 n-6 18:3 n-3 0 Delta 15 (pYX242) (9.27%) (0%) 18:3 n-6 18:4 n-3 0Delta 15 (9.18%) (0%) 20:3 n-6 20:4 n-3 0 Delta 17 (14.19%) (0%) 20:4n-6 20:5 n-3 0 Delta 17 (26.56%) (0%) 22:4 n-6 22:5 n-3 0 Delta 19(16.4%) (0%)${\%\mspace{14mu}{Conversion}} = {\frac{\left\lbrack {\%\mspace{14mu}{Product}} \right\rbrack}{\left\lbrack {{\%\mspace{14mu}{Product}} + {\%\mspace{14mu}{Substrate}}} \right\rbrack} \times 100}$*50 μM substrate used Numbers in parenthesis represent fatty acid as apercentage of total lipids from yeast. 18:2n-6 = Linoleic acid 18:3n-6 =gamma-linolenic acid 18:3n-3 = alpha-linolenic acid 18:4n-3 =stearidonic acid 20:5n-3 = eicosapentaenoic acid 20:3n-6 =dihomo-gamma-linolenic acid 20:4n-6 = arachidonic acid 22:4n-6 = adrenicacid 20:4n-3 = eicosatetraenoic acid 22:5n-3 = omega-3-docosapentaenoicacid

Table 3 displays the enzyme activity of the sdd17-encoded proteinproduct from Saprolegnia diclina (ATCC 56851). This enzyme is an activeomega-3 desaturase capable of desaturating both C₂₀ and C₂₂ omega-6fatty acids substrates to yield the corresponding omega-3 fatty acidproducts. This enzyme converted 13.8% of the added AA substrate to thecorresponding EPA product, thus indicating delta-17 desaturase activity.In addition, this enzyme also acted on DGLA, converting it to ETA, aswould be expected for a delta-17 desaturase. In this Example, however,only 5% of the added DGLA was converted to ETA, indicating that underthe conditions used here, the enzyme has a substrate preference for AAas compared to DGLA.

The activity of this enzyme toward C₂₂ fatty acid substrates was alsoinvestigated because C₂₂ omega-3 fatty acids like DPA and DHA haveimportant dietary and pharmaceutical implications. From Table 3, it canbe seen that this enzyme was active on C₂₂ substrates such as adrenicacid, converting 4% of it to DPA. As can be seen from the controlcultures, there was no non-specific conversion of exogenously addedsubstrate to product in non-transformed S. cerevisiae.

Table 4 demonstrates that this omega-3 desaturase (SDD17) can alsofunction at a lower temperature. (i.e. 15° C.). Here, 50 μM of exogenoussubstrate was added to the transformants and the cultures were grown for48 hours at 15° C. Fatty acid analysis was carried out as describedabove. The overall uptake of substrate by S. cerevisiae at 15° C. waslower than that seen at 24° C. (compare Table 3 & Table 4). However thepercent conversion of substrate to product by the enzyme increased at15° C. Since the presence of lower concentration of exogenous fatty acidsubstrate seemed to improve the activity of the enzyme, it is possiblethat fatty acid substrates at high concentrations may exert a feed-backinhibition on this enzyme. Further studies may be carried out todetermine the effect of different substrate concentration and differenttemperatures on expression of this omega-3 desaturase in S. cerevisiae.

TABLE 4 Yeast Expression of the Omega-3 Desaturase (SDD17) fromSaprolegnia diclina at 15° C. Substrate* Product % Enzyme CloneIncorporated Produced Conversion Activity PRSP19 18:2 n-6 18:3 n-3 0Delta 15 (8.79%) (0%) 18:3 n-6 18:4 n-3 0 Delta 15 (12.69%) (0%) 20:3n-6 20:4 n-3  13% Delta 17 (9.52%) (1.46%) 20:4 n-6 20:5 n-3  19% Delta17 (8.69%) (2.05%) 22:4 n-6 22:5 n-3 8.4% Delta 19 (6.68%) (0.62%)Control 18:2 n-6 18:3 n-3 0 Delta 15 (pYX242) (9.65%) (0%) 18:3 n-6 18:4n-3 0 Delta 15 (13.55%) (0%) 20:3 n-6 20:4 n-3 0 Delta 17 (10.17%) (0%)20:4 n-6 20:5 n-3 0 Delta 17 (16.58%) (0%) 22:4 n-6 22:5 n-3 0 Delta 19(11.05%) (0%) *50 μM substrate used Numbers in parenthesis representfatty acid as a percentage of total lipids from yeast 18:2 n-6 =Linoleic acid 20:3 n-6 = dihomo-gamma-linolenic acid 18:3 n-6 =gamma-linolenic acid 20:4 n-6 = arachidonic acid 18:3 n-3 =alpha-linolenic acid 22:4 n-6 = adrenic acid 18:4 n-3 = stearidonic acid20:4 n-3 = eicosatetraenoic acid 20:5 n-3 = eicosapentaenoic acid 22:5n-3 = omega-3 docosapentaenoic acidUnlike all known omega-3 desaturases, the sdd17-encoding enzyme did notdesaturate any C₁₈ omega-6 fatty acyl substrates to their correspondingomega-3 fatty acids (under the conditions tested). It is possible thatin vivo, this enzyme functions exclusively on AA, converting it to EPA.This would be consistent with the fatty acid profile of S. diclinadisplaying high amounts of AA and EPA, but little or none of the otheromega-3 intermediates (Table 5).

TABLE 5 Fatty Acid Profile of Saprolegnia diclina ATCC 56851 Fatty Acid% Total Lipid C10:0 0.22 C12:0 0.1 C13:0 3.98 C14:0 6.0 C14:1 n-5 0.29C15:0 1.06 C16:0 19.75 C16:1 n-7 1.99 C18:0 3.99 C18: n-9 15.39 C18:1n-7 7.39 C18:1 n-5 0.43 C18:2 n-6 7.07 C18:3 n-6 2.13 C18:3 n-3 0.08C20:0 0.76 C20:1 n-9 0.15 C20:1 n-7 0.08 C20:2 n-6 0.22 C20:3 n-6 1.31C20:4 n-6 15.42 C20:5 n-3 12.2

Thus, sdd17 encodes a novel omega-3 desaturase, capable of desaturatingC₂₀ and C₂₂ fatty acid substrates. The SDD17 protein can easily beexpressed in a heterologous system and thus has potential for use inother heterologous systems like plants. This enzyme is very differentfrom other known omega-3 desaturases, showing activity on both C₂₀ andC₂₂ fatty acid substrates, but not C₁₈ substrates. It shares only 31.6%identity with FAT-1, the only other known desaturase capable of actingon C₂₀ and C₂₂ omega-6 fatty acid substrates. Thus the enzyme encoded bysdd17 is a novel omega-3 desaturase.

EXAMPLE 6 Co-Expression of S. diclina Omega-3 Desaturase with OtherEnzymes

The pRSP20 construct consisting of sdd17 cloned into pYES2 yeastexpression vector, as described in Example 3, was used in co-expressionstudies with other desaturases and elongases. pRSP3, a construct thatcontained the delta 5-desaturase gene (SEQ ID NO: 27) from S. diclinacloned into the pYX242 yeast expression vector, was co-transformed withpRSP20 into yeast. Transformation protocol was as described in Example4. This delta 5-desaturase catalyzes the conversion of DGLA to AA andETA to EPA. Co-transformants were selected on minimal media lackingleucine and Uracil (DOB [-Leu-Ura]).

Table 6 shows that when 50 μM of the substrate DGLA (20:3 n-6) wasadded, the delta 5-desaturase converted it to AA (20:4, n-6), and theomega-3 desaturase was able to further desaturate AA to EPA (24:5, n-3).The percent conversion of the substrate to final product was 5%, with nobackground observed in the negative control.

TABLE 6 Co-expression Studies with the Omega -3 Desaturase (SDD17) fromS. diclina 20:3 n-6 20:4 n-6 20:5 n-3 Plasmid in (DGLA) (AA) (EPA) %Clone yeast Incorporated Produced Produced Conversion Cntrl pYX242 +19.33 0 0 0 pYES2 pRSP22 pRSP3 (Delta 20.56 2.64 0.14 5% 5) + pRSP20(omega-3 desaturase) 18:3 n-6 20:3 n-6 20:4 n-3 Plasmid in (GLA) (DGLA)(ETA) % Clone yeast Incorporated Produced Produced Conversion CntrlpYX242 + 4.83 0 0 0 pYES2 pRSP23 pRAT-4-A7 4.56 9.30 0.14 1.4%(elongase) + pRSP20 (omega-3 desaturase) * 50 μM substrate used Numbersrepresent fatty acid as a percentage of total lipids from yeast 18:3n-6= gamma-linolenic acid 20:3n-6 = dihomo-gamma-linolenic acid 20:4n-6 =arachidonic acid 20:4n-3 = eicosatetraenoic acid 20:5n-3 =eicosapentaenoic acid${\%\mspace{14mu}{Conversion}} = \frac{\left\lbrack {{\%\mspace{14mu}{Product}\mspace{14mu} 1} + {\%\mspace{14mu}{Product}\mspace{14mu} 2}} \right\rbrack}{\left\lbrack {{\%\mspace{14mu}{substrate}} + {\%\mspace{14mu}{Product}\mspace{14mu} 1} + {\%\mspace{20mu}{Product}\mspace{14mu} 2}} \right\rbrack}$

Table 6 also shows the results of a co-transformation experimentinvolving pRSP20 and pRAT-4-A7, an elongase obtained fromThraustochytrid sp. 7091 (SEQ. ID. NO: 28) cloned into pYX242. Thiselongase gene catalyzes the addition of two more carbons to thepre-existing PUFA. When 50 μM of the substrate GLA (18:3 n-6) was addedto the co-transformants, the elongase elongated GLA to DGLA, and theDGLA was further desaturated by the omega-3 desaturase to ETA (20:4n-3). The percent conversion of substrate to final product was 1.4%,with no background observed in the negative control.

Thus the S. diclina omega-3 desaturase was able to utilize a productproduced, in a heterologous expression system, by another heterologousenzyme from the PUFA biosynthetic pathway, and convert that product tothe expected PUFA.

It should be noted that the expression (and hence the activity) of sdd17when cloned in the pYES2 vector (pRSP20) was much lower than when clonedinto the pYX242 vector (pRSP19). This could be explained by thedifference in the expression promoters present in each vector. ThepYX242 promoter is a constitutive promoter and is much stronger than thegalactose-inducible promoter in pYES2. Similar observations have beenmade during expression studies with other desaturases when cloned intothese two expression vectors.

Further co-expression studies may be carried out using pRSP19 instead ofpRSP20 along with other desaturases and elongases. Also the S. diclinaomega-3 desaturase may also be co-expressed with other enzymes like thedelta 4-desaturase pRTA7 (SEQ. ID. NO: 29), where in adrenic acid (22:4n-G) may be added as a substrate and the final end product DHA (22:6n-3) may be produced due to the concerted action of the omega-3desaturase and the delta 4-desaturase.

EXAMPLE 7 Design of Degenerate Primers for the Isolation of the Delta12-Desaturase Gene from Saprolegnia diclina ATCC 56851

Analysis of the fatty acid composition of Saprolegnia diclina (ATCC56851) revealed the presence of a considerable amount of LA, whichsuggested the presence of a very active delta 12-desaturase (Table 5).Delta 12-desaturases use OA as a substrate, thus catalyzing theconversion of OA to LA (see FIG. 1). Delta 12-desaturases are presentonly in plants, fungi, and insects, but not in mammals, includinghumans. Thus LA is an “essential” fatty acid in humans because it cannotbe synthesized in vivo. LA is further desaturated and elongated toproduce important intracellular compounds like GLA, AA, and EPA.

The goal of this experiment was to isolate the delta 12-desaturase genefrom S. diclina and verify its functionality by expressing the enzyme ina heterologous host system such as yeast. The approach taken was todesign degenerate primers (oligonucleotides) that represent conservedamino acid motifs from known delta 12-desaturases. In designing theseprimers, known delta-12 desaturase sequence information from both fungiand plant sources was used, including sequence information from:Mortierella alpina (Accession #AF110509), Mucor rouxii (Accession#AF161219), Brassica juncea (Accession #X91139), Arabidopsis thaliana(Accession #L26296), and Borago officinalis (Accession #AF0744324). Thesequence information was analyzed using the CODEHOP Blockmaker program.

The degenerate primers used in this Example were as follows:

Protein Motif 1: NH₃— P N/E FTIKEIR D/E A/C IPAHCF —COOH Primer RO 967(Forward): 5′-CCG SAG TTC ACS ATC AAG GAG ATC (SEQ. ID. NO: 30) CGC GASKSC ATC CCG GCC CAC TGC TTC-3′. Protein Motif 2: NH₃— MP H/F YHAEEAT V/YH I/L KK A/L —COOH Primer RO 968 (Reverse): 5′-GRS CTT CTT GAK GTG GWMSGT GGC (SEQ. ID. NO: 31) CTC CTC GGC GTG GTA GWR CGG CAT-3′. ProteinMotif 3: NH₃— P L/V YW A/I C/M/A QG V/I V L/G/C TGVW —COOH Primer RO 964(Forward): 5′-CCS STC TAC TGG GCC TGC CAG GGT (SEQ. ID. NO: 32) RTC GTCCTC ACS GGT GTC TGG-3′.

This sequence is more similar to the known plant Delta 12-desaturases.

Primer RO 965 (Forward): 5′-CCS STC TAC TGG ATC RYS CAG GGT (SEQ. ID.NO: 33) RTC GTC KGY ACS GGT GTC TGG-3′.

This sequence is more similar to the known fungal Delta 12-desaturases.

Protein Motif 4: NH₃— HVAHH L/F FS T/Q MPHYHA —COOH (SEQ ID NO: 60)

Protein Motif 4: NH₃— HVAHH L/F FS T/Q MPHYHA —COOH Primer RO 966(Reverse): 5′-GGC GTG GTA GTG CGG CAT SMM CGA (SEQ. ID. NO: 34) GAA GARGTG GTG GGC GAC GTG-3′.

The degeneracy code used for the oligonucleotides was as follows: R=A/G;Y=C/T; M=A/C; K=G/T; W=A/T; S=C/G; B=C/G/T; D=A/G/T; H=A/C/T; V=A/C/G;N=A/C/G/T.

EXAMPLE 8 Identification and Isolation of the Delta 12-Desaturase Genefrom Saprolegnia diclina (ATCC 56851)

To isolate a fragment of the delta 12-desaturase gene from S. diclina,PCR was carried out using the S. diclina cDNA library from Example 1 asa template. Primers were used in the following combinations: (RO 964+RO966), (RO 965+RO 966), and (RO 967+RO 968). PCR was carried out in 100μl volumes using the “Taq PCR Master Mix”-brand polymerase (Qiagen). 10pmoles of each primer were used along with 1 μl of the cDNA library DNA.Amplification was carried out as follows: initial denaturation at 94° C.for 4 min, followed by 25 cycles of the following: 94° C. for 1 min, 47°C. for 1 min, 72° C. for 2 min. A final extension cycle of 72° C. for 5min was carried out, followed by reaction termination at 4° C.

Amplification with (RO 964+RO 966) or (RO 965+RO 966) resulted indistinct bands of ˜688 bp in length. Amplification with (RO 967+RO 968)resulted in one distinct band of ˜660 bp. These bands were resolved on a1% “SeaKem Gold”-brand agarose gel (FMC BioProducts), and gel-purifiedusing the Qiagen Gel Extraction Kit. The staggered ends on the fragmentwere “filled-in” using T4 DNA polymerase (Life Technologies), followingthe manufacturer's specifications. The DNA fragments were then clonedinto the PCR-Blunt vector (Invitrogen). The recombinant plasmids weretransformed into TOP10 supercompetent cells (Invitrogen), clones weresequenced, and a database search (Gen-EMBL) was carried out.

Clones “sdd12-1-8,” “sdd12-2-8,” and “sdd12-5-1” were all found tooverlap with each other, and these overlapping fragments were alignedusing the “ASSEMBLE”-brand program (GCG) to create a single fusionfragment of ˜900 bp. A “tFastA” search with the deduced amino acids ofthis fusion sequence showed highest identity to the following proteins:

49% identity in 298 amino acid overlap with Borago officinalis Delta12-desaturase (Accession # AF074324) and 46.7% identity in 332 aminoacid overlap with Sesamum indicum Delta 12-desaturase (Accession #AF192486).

Based on the high identity to known delta 12-desaturases, the fragmentwas considered to be a region of the S. diclina delta 12-desaturasegene. This fragment was used to design primers to pull up the 5′- and3′-ends of the gene.

To isolate the 3′ end of the gene, the following oligonucleotides weredesigned:

RO 975 (Forward): 5′-CAC GTA CCT CCA GCA CAC GGA CAC CTA CG-3′. (SEQ.ID. NO: 35) RO 976 (Forward): 5′-GAT CGA CAG CGC GAT CCA CCA CAT TGC-3′.(SEQ. ID. NO: 36)

These were used in combinations with the pBluescript SK(+) vectoroligonucleotide RO 898: 5′-CCC AGT CAC GAC GTT GTA AAA CGA CGG CCA G-3′(SEQ. ID. NO: 16).

PCR amplifications were carried out using “Taq PCR Master Mix”-brandpolymerase (Qiagen) as follows: 10 pmoles of each primer were used alongwith 1 μl of the cDNA library DNA from Example 1 as template.Amplification conditions were as follows: initial denaturation at 94° C.for 3 min, followed by 35 cycles of the following: 94° C. for 1 min, 60°C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for 7min was carried out, followed by reaction termination at 4° C.

Primer combination (RO 898+RO 975) generated a PCR band of ˜390 bp andprimer combination (RO 898+RO 976) generated a band of length ˜300 bp.These bands were purified and cloned into PCR-Blunt vector as describedearlier. Several clones, including clones “sdd12-8-14” and “sdd12-9-4”were found to contain the 3′ end of the delta 12-desaturase gene. Thesesequences overlapped the initial delta-12 desaturase fragment andincluded a TAA stop codon and a poly-A tail. Sequence analysis with the“tFastA” program revealed that clone “sdd12-9-4” shared 54.5% identityin a 73 amino acid overlap with the delta 12-desaturase from Mortierellaalpina (Accession #AB020033), and 56.9% identity in a 72 amino acidoverlap with the delta 12-desaturase from Mucor rouxii (Accession#AF161219).

To isolate the 5′ end of the this gene, the following oligonucleotideswere designed and used in combinations with the pBluescript SK(+) vectoroligonucleotide RO 899 (SEQ. ID. NO: 17).

RO 977 (Reverse): 5′-CAA ATG GTA AAA GCT AGT GGC AGC GCT GC-3′. (SEQ IDNO: 37) RO 978 (Reverse): 5′-AGT ACG TGC CCT GGA CGA ACC AGT AGA TG-3′.(SEQ ID NO: 38)

PCR amplifications were carried out using either “Taq PCR MasterMix”-brand polymerase (Qiagen) or the “Advantage-GC”-brand cDNA PCR kit(Clonetech). The Clonetech product was used to circumvent potential PCRamplification problems that may occur with GC-rich regions generallypresent at the 5′-end of desaturases from this organism. PCRamplifications using the “Taq PCR Master Mix”-brand polymerase wascarried out as described for the isolation of 3′-end of this gene.

When using the “Advantage-GC cDNA PCR”-brand kit, thermocyclingconditions were as follows: the template was initially denatured at 94°C. for 1 min, followed by 30 cycles of 94° C. for 30 seconds, 68° C. for3 minutes, and finally an extension cycle at 68° C. for 5 min. Eachreaction included 1 μl of cDNA library template (from Example 1), 10pmole of each primer, 0.2 mM each dNTP, 1M GC Melt, 40 mM Tricine-KOH,15 mM KOAc, 3.5 mM MG(OAc)₂, 5% DMSO, and 375 μg/ml BSA in a totalvolume of 50 μl.

A PCR product of ˜371 bp was obtained using the primer combination (RO899+RO 978). This band was cloned into the PCR-Blunt vector (Invitrogen)as described earlier. Only one clone, “sdd12-10-8,” thus obtainedcontained the putative ATG start site of the gene. Other clones had theATG replaced by other codons. “tFastA” analysis of the deduced aminoacid sequence of “sdd12-10-8” showed 47.2% identity in a 72 amino acidoverlap of the delta-12 desaturase from Impatiens balsamina (Accession#AF182520) and 42.7% identity in a 75 amino acid overlap with thedelta-12 desaturase from Calendula officinalis (Accession #AJ245938).

The full length of this delta 12-desaturase was obtained by PCRamplification of the S. diclina cDNA library of Example 1 using thefollowing oligonucleotides:

RO1051 (Forward): 5′-TCA ACA GAA TTC ATG TGC AAA GGT CAA GCT CCT TCC AAGGCC GAC GTG-3′ (SEQ. ID. 39). This primer contains the ‘ATG’ start site(underlined) followed by the 5′ sequence of the Delta 12-desaturase. Inaddition, an EcoRI site (double underline) was introduced upstream ofthe start site to facilitate cloning into the pYX242 yeast expressionvector.

RO1057 (Reverse): 5′-AAA AGA AAG CTT TTA CTT TTC CTC GAG CTT GCG CTT GTAAAA CAC AAC-3′ (SEQ. ID. NO: 40). This primer contains the TAA stopcodon (underlined) of the gene as well as a HindIII site (doubleunderline), which was included for convenient cloning into the pYX242yeast expression vector.

PCR amplifications were carried out using both “Taq PCR MasterMix”-brand polymerase (Qiagen) and the “Advantage-GC cDNA PCR”-brand kit(Clonetech), as described earlier. In this case, however, S. diclinagenomic DNA was used as the template for amplification. A PCR band of˜1.1 kb was thus obtained and this band was isolated, purified, clonedinto PCR-Blunt vector (Invitrogen), and transformed into TOP10 cells.The inserts were sequenced to verify the gene sequence. Clone“sdd12-gg-b1” was digested with EcoRI/HindIII to release the full-lengthinsert, and this insert was cloned into the yeast expression vectorpYX242, previously digested with EcoRI/HindIII. This construct included1182 bp of the delta-12 desaturase gene and pYX242. The construct waslabeled pRSP11. The pRSP11 construct was then transformed into S.cerevisiae (SC334) for expression studies.

The full-length gene of this putative delta-12 desaturase (labeledsdd12) was 1182 bp in length (SEQ ID NO: 41) (FIG. 6). The gene encodesa protein of 393 amino acid residues (SEQ ID NO: 42) (FIG. 7). A“tFastA” search of the deduced protein sequence of sdd12 showed theprotein to have highest identity (45.6% in a 379 amino acid overlap)with the delta-12 desaturase from Gossypium hirsutum (Accession #X97016)(FIG. 8) and 49.6% identity in a 353 amino acid overlap with thedelta-12 desaturase from Sesamum indicum (FAD2) (Accession #AF192486).

Like other delta-12 desaturases, this enzyme also does not contain acytochrome b5 domain within the 5′ end of its' sequence. This enzymedoes contain the three histidine-rich sequences that are present in allmembrane-bound desaturases. The position and length of thesehistidine-boxes are similar to those seen in other desaturases. Theseare present at amino acid positions 108 to 112 (HECGH), 144 to 148(HRRHH), and 326 to 330 (HVTHH) of SEQ. ID. NO: 42. As noted earlier,these histidine-rich boxes are believed to co-ordinate the diiron-oxostructure at the enzyme's active site and are necessary for enzymeactivity.

EXAMPLE 9 Expression of the Delta 12-Desaturase Gene (sdd12) in Bakers'Yeast

To determine the substrate specificity and the class of reactioncatalyzed by this delta 12-desaturase (SDD12), sdd12 was heterologouslyexpressed in a Saccharomyces cerevisiae (SC334). As noted earlier,because S. cerevisiae cannot synthesize fatty acids beyond OA, it is anideal system to determine enzyme activity on substrates longer than OAbecause no background enzyme activity will be detected. Suitable fattyacid substrates are exogenously supplied to the host; these substratesare taken up by the cell and acted on by the expressed delta-12desaturase of the transformed sdd12 gene.

Clone pRSP11, which contained the full-length delta-12 desaturase(sdd12) from S. diclina, cloned into pYX242, was transformed intoSaccharomyces cerevisiae (SC334) using the “Alkali-Cation YeastTransformation”-brand kit (BIO 101), following the manufacturer'sinstructions. Transformants were selected for leucine auxotrophy onmedia lacking leucine (DOB-Leu). To detect the specific desaturaseactivity of these clones, transformants were grown in the presence of 50μM each of OA, LA, GLA, and DGLA.

Conversion of OA to LA (18:2n-6) indicates delta-12 desaturase activity.

Conversion of LA to ALA (18:3n-3) indicates delta-15 desaturaseactivity.

Conversion of LA to GLA (18:3n-6) indicates delta-6 desaturase activity.

-   -   Conversion of GLA to stearidonic (18:4n-3) acid indicates        delta-15 desaturase activity.    -   Conversion of DGLA to ETA (20:4n-3) indicates delta-17        desaturase activity.    -   Conversion of DGLA to AA (20:4n-6) indicates delta-5 desaturase        activity.

The negative control strain was S. cerevisiae transformed with thepYX242 vector. The experimental and control cultures were grownsimultaneously and analyzed.

The cultures were vigorously agitated (250 rpm) and grown for 48 hoursat 24° C. in the presence of 50 μM (final concentration) of the varioussubstrates (see Table 7). The cells were spun down, washed once indistilled water, and the pellets resuspended in methanol; chloroform wasadded along with tritridecanoin (as an internal standard). Thesemixtures were incubated for at least an hour at room temperature, or at4° C. overnight. The chloroform layer was extracted and filtered througha Whatman filter with 1 gm anhydrous sodium sulfate to removeparticulate matter and residual water. The organic solvents wereevaporated at 40° C. under a stream of nitrogen. The extracted lipidswere then converted to fatty acid methyl esters (FAME) for gaschromatography analysis (GC) by adding 2 ml 0.5 N potassium hydroxide inmethanol to a closed tube. The samples were heated to 95° C.–100° C. for30 minutes and cooled to room temperature. Approximately 2 ml 14%borontrifluoride in methanol was added and the heating repeated. Afterthe extracted lipid mixture cooled, 2 ml of water and 1 ml of hexanewere added to extract the FAME for analysis by GC. The percentconversion was calculated using the formula:

${\%\mspace{14mu}{Conversion}} = {\frac{\left\lbrack {\%\mspace{14mu}{Product}} \right\rbrack}{\left\lbrack {{\%\mspace{11mu}{Product}} + {\%\mspace{14mu}{Substrate}}} \right\rbrack} \times 100}$

Table 7 shows the enzyme activity of the delta-12 desaturase whenexpressed in yeast. Here, the pRSP11 clone when expressed was capable ofconverting 35.8% of OA substrate to LA, indicating delta-12 desaturaseactivity.

In Table 7, the fatty acids of interest are represented as a percentageof the total lipids extracted from yeast. GC/MS was employed to identifythe products. Under these conditions, the clones did not exhibit otherdesaturase activities. This confirmed that the gene isolated is adelta-12 desaturase gene. No background substrate conversion wasdetected using hosts transformed with just the vector alone. This dataindicates that this delta-12 desaturase can be expressed in aheterologous system and is thus useful in the production of transgenicpolyunsaturated fatty acids like GLA, AA, EPA and DHA.

TABLE 7 Saprolegnia diclina Delta 12-Desaturase Expression in Baker'sYeast at 24° C. % Desatur. Substrate* Product Conversion Clone ActivityIncorporated Produced of Substrate pRSP11 Delta OA (17.09%) LA (9.59%)35.8% (pYX242 + 12 Delta 12- Delta LA (18.14%) ALA (0.06%) 0 Desaturase15 (S. Delta LA (18.14%) GLA (0%) 0 diclina)) 6 Delta 5 DGLA (25.38%) AA(0.17%) 0 Delta DGLA (25.38%) ETA (0.07%) 0 17 Control Delta OA (18.99%)LA (0.09%) 0 (pYX242) 12 Delta LA (8.63%) ALA (0%) 0 15 Delta LA (8.63%)GLA (0%) 0 6 Delta DGLA (13.74%) AA (0%) 0 5 Delta DGLA (13.74%) ETA(0%) 0 17 *50 μM substrate used Numbers in parenthesis represent fattyacid as a percentage of total lipids from yeast.Nutritional Compositions

The PUFAs described in the Detailed Description may be utilized invarious nutritional supplements, infant formulations, nutritionalsubstitutes and other nutritional solutions.

I. Infant Formulations

A. Isomil® Soy Formula with Iron:

-   Usage: As a beverage for infants, children and adults with an    allergy or sensitivity to cows milk. A feeding for patients with    disorders for which lactose should be avoided: lactase deficiency,    lactose intolerance and galactosemia.-   Features:    -   Soy protein isolate to avoid symptoms of cow's-milk-protein        allergy or sensitivity.    -   Lactose-free formulation to avoid lactose-associated diarrhea.    -   Low osmolality (240 mOs/kg water) to reduce risk of osmotic        diarrhea.    -   Dual carbohydrates (corn syrup and sucrose) designed to enhance        carbohydrate absorption and reduce the risk of exceeding the        absorptive capacity of the damaged gut.    -   1.8 mg of Iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Recommended levels of vitamins and minerals.    -   Vegetable oils to provide recommended levels of essential fatty        acids.    -   Milk-white color, milk-like consistency and pleasant aroma.-   Ingredients: (Pareve) 85% water, 4.9% corn syrup, 2.6% sugar    (sucrose), 2.1% soy oil, 1.9% soy protein isolate, 1.4% coconut oil,    0.15% calcium citrate, 0.11% calcium phosphate tribasic, potassium    citrate, potassium phosphate monobasic, potassium chloride, mono-    and disglycerides, soy lecithin, carrageenan, ascorbic acid,    L-methionine, magnesium chloride, potassium phosphate dibasic,    sodium chloride, choline chloride, taurine, ferrous sulfate,    m-inositol, alpha-tocopheryl acetate, zinc sulfate, L-carnitine,    niacinamide, calcium pantothenate, cupric sulfate, vitamin A    palmitate, thiamine chloride hydrochloride, riboflavin, pyridoxine    hydrochloride, folic acid, manganese sulfate, potassium iodide,    phylloquinone, biotin, sodium selenite, vitamin D3 and    cyanocobalamin.    B. Isomil® DF Soy Formula for Diarrhea:-   Usage: As a short-term feeding for the dietary management of    diarrhea in infants and toddlers.-   Features:    -   First infant formula to contain added dietary fiber from soy        fiber specifically for diarrhea management.    -   Clinically shown to reduce the duration of loose, watery stools        during mild to severe diarrhea in infants.    -   Nutritionally complete to meet the nutritional needs of the        infant.    -   Soy protein isolate with added L-methionine meets or exceeds an        infant's requirement for all essential amino acids.    -   Lactose-free formulation to avoid lactose-associated diarrhea.    -   Low osmolality (240 mOsm/kg water) to reduce the risk of osmotic        diarrhea.    -   Dual carbohydrates (corn syrup and sucrose) designed to enhance        carbohydrate absorption and reduce the risk of exceeding the        absorptive capacity of the damaged gut.    -   Meets or exceeds the vitamin and mineral levels recommended by        the Committee on Nutrition of the American Academy of Pediatrics        and required by the Infant Formula Act.    -   1.8 mg of iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Vegetable oils to provide recommended levels of essential fatty        acids.-   Ingredients: (Pareve) 86% water, 4.8% corn syrup, 2.5% sugar    (sucrose), 2.1% soy oil, 2.0% soy protein isolate, 1.4% coconut oil,    0.77% soy fiber, 0.12% calcium citrate, 0.11% calcium phosphate    tribasic, 0.10% potassium citrate, potassium chloride, potassium    phosphate monobasic, mono and diglycerides, soy lecithin,    carrageenan, magnesium chloride, ascorbic acid, L-methionine,    potassium phosphate dibasic, sodium chloride, choline chloride,    taurine, ferrous sulfate, m-inositol, alpha-tocopheryl acetate, zinc    sulfate, L-carnitine, niacinamide, calcium pantothenate, cupric    sulfate, vitamin A palmitate, thiamine chloride hydrochloride,    riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate,    potassium iodide, phylloquinone, biotin, sodium selenite, vitamin D3    and cyanocobalamin.    C. Isomil® SF Sucrose-Free Soy Formula with Iron:-   Usage: As a beverage for infants, children and adults with an    allergy or sensitivity to cow's-milk protein or an intolerance to    sucrose. A feeding for patients with disorders for which lactose and    sucrose should be avoided.-   Features:    -   Soy protein isolate to avoid symptoms of cow's-milk-protein        allergy or sensitivity.    -   Lactose-free formulation to avoid lactose-associated diarrhea        (carbohydrate source is Polycose® Glucose Polymers).    -   Sucrose free for the patient who cannot tolerate sucrose.    -   Low osmolality (180 mOsm/kg water) to reduce risk of osmotic        diarrhea.    -   1.8 mg of iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Recommended levels of vitamins and minerals.    -   Vegetable oils to provide recommended levels of essential fatty        acids.    -   Milk-white color, milk-like consistency and pleasant aroma.-   Ingredients: (Pareve) 75% water, 11.8% hydrolized cornstarch, 4.1%    soy oil, 4.1% soy protein isolate, 2.8% coconut oil, 1.0% modified    cornstarch, 0.38% calcium phosphate tribasic, 0.17% potassium    citrate, 0.13% potassium chloride, mono- and diglycerides, soy    lecithin, magnesium chloride, abscorbic acid, L-methionine, calcium    carbonate, sodium chloride, choline chloride, carrageenan, taurine,    ferrous sulfate, m-inositol, alpha-tocopheryl acetate, zinc sulfate,    L-carnitine, niacinamide, calcium pantothenate, cupric sulfate,    vitamin A palmitate, thiamine chloride hydrochloride, riboflavin,    pyridoxine hydrochloride, folic acid, manganese sulfate, potassium    iodide, phylloquinone, biotin, sodium selenite, vitamin D3 and    cyanocobalamin.    D. Isomil® 20 Soy Formula with Iron Ready to Feed, 20 Cal/fl oz.:-   Usage: When a soy feeding is desired.-   Ingredients: (Pareve) 85% water, 4.9% corn syrup, 2.6%    sugar(sucrose), 2.1% soy oil, 1.9% soy protein isolate, 1.4% coconut    oil, 0.15% calcium citrate, 0.11% calcium phosphate tribasic,    potassium citrate, potassium phosphate monobasic, potassium    chloride, mono- and diglycerides, soy lecithin, carrageenan,    abscorbic acid, L-methionine, magnesium chloride, potassium    phosphate dibasic, sodium chloride, choline chloride, taurine,    ferrous sulfate, m-inositol, alpha-tocopheryl acetate, zinc sulfate,    L-carnitine, niacinamide, calcium pantothenate, cupric sulfate,    vitamin A palmitate, thiamine chloride hydrochloride, riboflavin,    pyridoxine hydrochloride, folic acid, manganese sulfate, potassium    iodide, phylloquinone, biotin, sodium selenite, vitamin D3 and    cyanocobalamin.    E. Similac® Infant Formula:-   Usage: When an infant formula is needed: if the decision is made to    discontinue breastfeeding before age 1 year, if a supplement to    breastfeeding is needed or as a routine feeding if breastfeeding is    not adopted.-   Features:    -   Protein of appropriate quality and quantity for good growth;        heat-denatured, which reduces the risk of milk-associated        enteric blood loss.    -   Fat from a blend of vegetable oils (doubly homogenized),        providing essential linoleic acid that is easily absorbed.    -   Carbohydrate as lactose in proportion similar to that of human        milk.    -   Low renal solute load to minimize stress on developing organs.    -   Powder, Concentrated Liquid and Ready To Feed forms.-   Ingredients: (-D) Water, nonfat milk, lactose, soy oil, coconut oil,    mono- and diglycerides, soy lecithin, abscorbic acid, carrageenan,    choline chloride, taurine, m-inositol, alpha-tocopheryl acetate,    zinc sulfate, niacinamide, ferrous sulfate, calcium pantothenate,    cupric sulfate, vitamin A palmitate, thiamine chloride    hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid,    manganese sulfate, phylloquinone, biotin, sodium selenite, vitamin    D3 and cyanocobalamin.    F. Similac® NeoCare Premature Infant Formula with Iron:-   Usage: For premature infants' special nutritional needs after    hospital discharge. Similac NeoCare is a nutritionally complete    formula developed to provide premature infants with extra calories,    protein, vitamins and minerals needed to promote catch-up growth and    support development.-   Features:    -   Reduces the need for caloric and vitamin supplementation. More        calories (22 Cal/fl oz) than standard term formulas (20 Cal/fl        oz).    -   Highly absorbed fat blend, with medium-chain triglycerides        (MCToil) to help meet the special digestive needs of premature        infants.    -   Higher levels of protein, vitamins and minerals per 100 calories        to extend the nutritional support initiated in-hospital.    -   More calcium and phosphorus for improved bone mineralization.-   Ingredients: -D Corn syrup solids, nonfat milk, lactose, whey    protein concentrate, soy oil, high-oleic safflower oil, fractionated    coconut oil (medium chain triglycerides), coconut oil, potassium    citrate, calcium phosphate tribasic, calcium carbonate, ascorbic    acid, magnesium chloride, potassium chloride, sodium chloride,    taurine, ferrous sulfate, m-inositol, choline chloride, ascorbyl    palmitate, L-carnitine, alpha-tocopheryl acetate, zinc sulfate,    niacinamide, mixed tocopherols, sodium citrate, calcium    pantothenate, cupric sulfate, thiamine chloride hydrochloride,    vitamin A palmitate, beta carotene, riboflavin, pyridoxine    hydrochloride, folic acid, manganese sulfate, phylloquinone, biotin,    sodium selenite, vitamin D3 and cyanocobalamin.    G. Similac Natural Care Low-Iron Human Milk Fortifier Ready to Use,    24 Cal/fl oz.:-   Usage: Designed to be mixed with human milk or to be fed    alternatively with human milk to low-birth-weight infants.-   Ingredients: -D Water, nonfat milk, hydrolyzed cornstarch, lactose,    fractionated coconut oil (medium-chain triglycerides), whey protein    concentrate, soy oil, coconut oil, calcium phosphate tribasic,    potassium citrate, magnesium chloride, sodium citrate, ascorbic    acid, calcium carbonate, mono and diglycerides, soy lecithin,    carrageenan, choline chloride, m-inositol, taurine, niacinamide,    L-carnitine, alpha tocopheryl acetate, zinc sulfate, potassium    chloride, calcium pantothenate, ferrous sulfate, cupric sulfate,    riboflavin, vitamin A palmitate, thiamine chloride hydrochloride,    pyridoxine hydrochloride, biotin, folic acid, manganese sulfate,    phylloquinone, vitamin D3, sodium selenite and cyanocobalamin.

Various PUFAs of this invention can be substituted and/or added to theinfant formulae described above and to other infant formulae known tothose in the art.

II. Nutritional Formulations

A. ENSURE®

-   Usage: ENSURE is a low-residue liquid food designed primarily as an    oral nutritional supplement to be used with or between meals or, in    appropriate amounts, as a meal replacement. ENSURE is lactose- and    gluten-free, and is suitable for use in modified diets, including    low-cholesterol diets. Although it is primarily an oral supplement,    it can be fed by tube.-   Patient Conditions:    -   For patients on modified diets    -   For elderly patients at nutrition risk    -   For patients with involuntary weight loss    -   For patients recovering from illness or surgery    -   For patients who need a low-residue diet-   Ingredients: -D Water, Sugar (Sucrose), Maltodextrin (Corn), Calcium    and Sodium Caseinates, High-Oleic Safflower Oil, Soy Protein    Isolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium Phosphate    Tribasic, Sodium Citrate, Magnesium Chloride, Magnesium Phosphate    Dibasic, Artificial Flavor, Sodium Chloride, Soy Lecithin, Choline    Chloride, Ascorbic Acid, Carrageenan, Zinc Sulfate, Ferrous Sulfate,    Alpha-Tocopheryl Acetate, Gellan Gum, Niacinamide, Calcium    Pantothenate, Manganese Sulfate, Cupric Sulfate, Vitamin A    Palmitate, Thiamine Chloride Hydrochloride, Pyridoxine    Hydrochloride, Riboflavin, Folic Acid, Sodium Molybdate, Chromium    Chloride, Biotin, Potassium Iodide, Sodium Selenate.    B. ENSURE® BARS:-   Usage: ENSURE BARS are complete, balanced nutrition for supplemental    use between or with meals. They provide a delicious, nutrient-rich    alternative to other snacks. ENSURE BARS contain <1 g lactose/bar,    and Chocolate Fudge Brownie flavor is gluten-free. (Honey Graham    Crunch flavor contains gluten.)-   Patient Conditions:    -   For patients who need extra calories, protein, vitamins and        minerals.    -   Especially useful for people who do not take in enough calories        and nutrients.    -   For people who have the ability to chew and swallow    -   Not to be used by anyone with a peanut allergy or any type of        allergy to nuts.-   Ingredients: Honey Graham Crunch—High-Fructose Corn Syrup, Soy    Protein Isolate, Brown Sugar, Honey, Maltodextrin (Corn), Crisp Rice    (Milled Rice,    -   Sugar [Sucrose], Salt [Sodium Chloride] and Malt), Oat Bran,        Partially Hydrogenated Cottonseed and Soy Oils, Soy        Polysaccharide, Glycerine, Whey Protein Concentrate,        Polydextrose, Fructose, Calcium Caseinate, Cocoa Powder,        Artificial Flavors, Canola Oil, High-Oleic Safflower Oil, Nonfat        Dry Milk, Whey Powder, Soy Lecithin and Corn Oil. Manufactured        in a facility that processes nuts.-   Vitamins and Minerals: Calcium Phosphate Tribasic, Potassium    Phosphate Dibasic, Magnesium Oxide, Salt (Sodium Chloride),    Potassium Chloride, Ascorbic Acid, Ferric Orthophosphate,    Alpha-Tocopheryl Acetate, Niacinamide, Zinc Oxide, Calcium    Pantothenate, Copper Gluconate, Manganese Sulfate, Riboflavin, Beta    Carotene, Pyridoxine Hydrochloride, Thiamine Mononitrate, Folic    Acid, Biotin, Chromium Chloride, Potassium Iodide, Sodium Selenate,    Sodium Molybdate, Phylloquinone, Vitamin D3 and Cyanocobalamin.-   Protein: Honey Graham Crunch—The protein source is a blend of soy    protein isolate and milk proteins.

Soy protein isolate 74% Milk proteins 26%

-   Fat: Honey Graham Crunch—The fat source is a blend of partially    hydrogenated cottonseed and soybean, canola, high oleic safflower,    oils, and soy lecithin.

Partially hydrogenated cottonseed and soybean oil 76% Canola oil 8%High-oleic safflower oil 8% Corn oil 4% Soy lecithin 4%

-   Carbohydrate: Honey Graham Crunch—The carbohydrate source is a    combination of high-fructose corn syrup, brown sugar, maltodextrin,    honey, crisp rice, glycerine, soy polysaccharide, and oat bran.

High-fructose corn syrup 24% Brown sugar 21% Maltodextrin 12% Honey 11%Crisp rice 9% Glycerine 9% Soy Polysaccharide 7% Oat bran 7%C. ENSURE® HIGH PROTEIN:

-   Usage: ENSURE HIGH PROTEIN is a concentrated, high-protein liquid    food designed for people who require additional calories, protein,    vitamins, and minerals in their diets. It can be used as an oral    nutritional supplement with or between meals or, in appropriate    amounts, as a meal replacement. ENSURE HIGH PROTEIN is lactose- and    gluten-free, and is suitable for use by people recovering from    general surgery or hip fractures and by patients at risk for    pressure ulcers.-   Patient Conditions:    -   For patients who require additional calories, protein, vitamins,        and minerals, such as patients recovering from general surgery        or hip fractures, patients at risk for pressure ulcers, and        patients on low-cholesterol diets.-   Features:    -   Low in saturated fat    -   Contains 6 g of total fat and <5 mg of cholesterol per serving    -   Rich, creamy taste    -   Excellent source of protein, calcium, and other essential        vitamins and minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested-   Ingredients:-   Vanilla Supreme: -D Water, Sugar (Sucrose), Maltodextrin (Corn),    Calcium and Sodium Caseinates, High-OIeic Safflower Oil, Soy Protein    Isolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium Phosphate    Tribasic, Sodium Citrate, Magnesium Chloride, Magnesium Phosphate    Dibasic, Artificial Flavor, Sodium Chloride, Soy Lecithin, Choline    Chloride, Ascorbic Acid, Carrageenan, Zinc Sulfate, Ferrous Suffate,    Alpha-Tocopheryl Acetate, Gellan Gum, Niacinamide, Calcium    Pantothenate, Manganese Sulfate, Cupric Sulfate, Vitamin A    Palmitate, Thiamine Chloride Hydrochloride, Pyridoxine    Hydrochloride, Riboflavin, Folic Acid, Sodium Molybdate, Chromium    Chloride, Biotin, Potassium Iodide, Sodium Selenate, Phylloquinone,    Vitamin D3 and Cyanocobalamin.-   Protein:

The protein source is a blend of two high-biologic-value proteins:casein and soy.

Sodium and calcium caseinates 85% Soy protein isolate 15%

-   Fat:

The fat source is a blend of three oils: high-oleic safflower, canola,and soy.

High-oleic safflower oil 40% Canola oil 30% Soy oil 30%The level of fat in ENSURE HIGH PROTEIN meets American Heart Association(AHA) guidelines. The 6 grams of fat in ENSURE HIGH PROTEIN represent24% of the total calories, with 2.6% of the fat being from saturatedfatty acids and 7.9% from polyunsaturated fatty acids. These values arewithin the AHA guidelines of <30% of total calories from fat, <10% ofthe calories from saturated fatty acids, and <10% of total calories frompolyunsaturated fatty acids.

-   Carbohydrate:

ENSURE HIGH PROTEIN contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla supreme, chocolate royal,wild berry, and banana), plus VARI-FLAVORS® Flavor Pacs in pecan,cherry, strawberry, lemon, and orange, help to prevent flavor fatigueand aid in patient compliance.

-   Vanilla and Other Nonchocolate Flavors:

Sucrose 60% Maltodextrin 40% Chocolate: Sucrose 70% Maltodextrin 30%D. ENSURE® LIGHT

-   Usage: ENSURE LIGHT is a low-fat liquid food designed for use as an    oral nutritional supplement with or between meals. ENSURE LIGHT is    lactose- and gluten-free, and is suitable for use in modified diets,    including low-cholesterol diets.-   Patient Conditions:    -   For normal-weight or overweight patients who need extra        nutrition in a supplement that contains 50% less fat and 20%        fewer calories than ENSURE.    -   For healthy adults who do not eat right and need extra        nutrition.-   Features:    -   Low in fat and saturated fat    -   Contains 3 g of total fat per serving and <5 mg cholesterol    -   Rich, creamy taste    -   Excellent source of calcium and other essential vitamins and        minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested-   Ingredients:-   French Vanilla: -D Water, Maltodextrin (Corn), Sugar (Sucrose),    Calcium Caseinate, High-Oleic Safflower Oil, Canola Oil, Magnesium    Chloride, Sodium Citrate, Potassium Citrate, Potassium Phosphate    Dibasic, Magnesium Phosphate Dibasic, Natural and Artificial Flavor,    Calcium Phosphate Tribasic, Cellulose Gel, Choline Chloride, Soy    Lecithin, Carrageenan, Salt (Sodium Chloride), Ascorbic Acid,    Cellulose Gum, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Zinc    Sulfate, Niacinamide, Manganese Sulfate, Calcium Pantothenate,    Cupric Sulfate, Thiamine Chloride Hydrochloride, Vitamin A    Palmitate, Pyridoxine Hydrochloride, Riboflavin, Chromium Chloride,    Folic Acid, Sodium Molybdate, Biotin, Potassium Iodide, Sodium    Selenate, Phylloquinone, Vitamin D3 and Cyanocobalamin.-   Protein:    The protein source is calcium caseinate.

Calcium caseinate 100%Fat:The fat source is a blend of two oils: high-oleic safflower and canola.

High-oleic safflower oil 70% Canola oil 30%The level of fat in ENSURE LIGHT meets American Heart Association (AHA)guidelines. The 3 grams of fat in ENSURE LIGHT represent 13.5% of thetotal calories, with 1.4% of the fat being from saturated fatty acidsand 2.6% from polyunsaturated fatty acids. These values are within theAHA guidelines of <30% of total calories from fat, <10% of the, caloriesfrom saturated fatty acids, and <10% of total calories frompolyunsaturated fatty acids.

-   Carbohydrate:    ENSURE LIGHT contains a combination of maltodextrin and sucrose. The    chocolate flavor contains corn syrup as well. The mild sweetness and    flavor variety (French vanilla, chocolate supreme, strawberry    swirl), plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry,    lemon, and orange, help to prevent flavor fatigue and aid in patient    compliance.-   Vanilla and Other Nonchocolate Flavors:

Sucrose   51% Maltodextrin   49% Chocolate: Sucrose 47.0% Corn Syrup26.5% Maltodextrin 26.5%

-   Vitamins and Minerals:    An 8-fl-oz serving of ENSURE LIGHT provides at least 25% of the RDIs    for 24 key vitamins and minerals.-   Caffeine:

Chocolate flavor contains 2.1 mg caffeine/8 fl oz.

E. ENSURE PLUS®

-   Usage: ENSURE PLUS is a high-calorie, low-residue liquid food for    use when extra calories and nutrients, but a normal concentration of    protein, are needed. It is designed primarily as an oral nutritional    supplement to be used with or between meals or, in appropriate    amounts, as a meal replacement. ENSURE PLUS is lactose- and    gluten-free. Although it is primarily an oral nutritional    supplement, it can be fed by tube.-   Patient Conditions:    -   For patients who require extra calories and nutrients, but a        normal concentration of protein, in a limited volume.    -   For patients who need to gain or maintain healthy weight.-   Features:    -   Rich, creamy taste    -   Good source of essential vitamins and minerals-   Ingredients:-   Vanilla: -D Water, Corn Syrup, Maltodextrin (Corn), Corn Oil, Sodium    and Calcium Caseinates, Sugar (Sucrose), Soy Protein Isolate,    Magnesium Chloride, Potassium Citrate, Calcium Phosphate Tribasic,    Soy Lecithin, Natural and Artificial Flavor, Sodium Citrate,    Potassium Chloride, Choline Chloride, Ascorbic Acid, Carrageenan,    Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate,    Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric    Sulfate, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride,    Riboflavin, Vitamin A Palmitate, Folic Acid, Biotin, Chromium    Chloride, Sodium Molybdate, Potassium Iodide, Sodium Selenite,    Phylloquinone, Cyanocobalamin and Vitamin D3.-   Protein:

The protein source is a blend of two high-biologic-value proteins:casein and soy.

Sodium and calcium caseinates 84% Soy protein isolate 16%

-   Fat:

The fat source is corn oil.

Corn oil 100%

-   Carbohydrate:

ENSURE PLUS contains a combination of maltodextrin and sucrose. The mildsweetness and flavor variety (vanilla, chocolate, strawberry, coffee,buffer pecan, and eggnog), plus VARI-FLAVORS® Flavor Pacs in pecan,cherry, strawberry, lemon, and orange, help to prevent flavor fatigueand aid in patient compliance.

-   Vanilla, Strawberry, Butter Pecan, and Coffee Flavors:

Corn Syrup 39% Maltodextrin 38% Sucrose 23%

-   Chocolate and Eggnog Flavors:

Corn Syrup 36% Maltodextrin 34% Sucrose 30%

-   Vitamins and Minerals:

An 8-fl-oz serving of ENSURE PLUS provides at least 15% of the RDIs for25 key Vitamins and minerals.

-   Caffeine:    Chocolate flavor contains 3.1 mg Caffeine/8 fl oz. Coffee flavor    contains a trace amount of caffeine.    F. ENSURE PLUS® HN-   Usage: ENSURE PLUS HN is a nutritionally complete high-calorie,    high-nitrogen liquid food designed for people with higher calorie    and protein needs or limited volume tolerance. It may be used for    oral supplementation or for total nutritional support by tube.    ENSURE PLUS HN is lactose- and gluten-free.-   Patient Conditions:    -   For patients with increased calorie and protein needs, such as        following surgery or injury.    -   For patients with limited volume tolerance and early satiety.-   Features:    -   For supplemental or total nutrition    -   For oral or tube feeding    -   1.5 CaVmL,    -   High nitrogen    -   Calorically dense-   Ingredients:-   Vanilla: -D Water, Maltodextrin (Corn), Sodium and Calcium    Caseinates, Corn Oil, Sugar (Sucrose), Soy Protein Isolate,    Magnesium Chloride, Potassium Citrate, Calcium Phosphate Tribasic,    Soy Lecithin, Natural and Artificial Flavor, Sodium Citrate, Choline    Chloride, Ascorbic Acid, Taurine, L-Carnitine, Zinc Sulfate, Ferrous    Sulfate, Alpha-Tocopheryl Acetate, Niacinamide, Carrageenan, Calcium    Pantothenate, Manganese Sulfate, Cupric Sulfate, Thiamine Chloride    Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Vitamin A    Palmitate, Folic Acid, Biotin, Chromium Chloride, Sodium Molybdate,    Potassium Iodide, Sodium Selenite, Phylloquinone, Cyanocobalamin and    Vitamin D3.    G. ENSURE® POWDER:-   Usage: ENSURE POWDER (reconstituted with water) is a low-residue    liquid food designed primarily as an oral nutritional supplement to    be used with or between meals. ENSURE POWDER is lactose- and    gluten-free, and is suitable for use in modified diets, including    low-cholesterol diets.-   Patient Conditions:    -   For patients on modified diets    -   For elderly patients at nutrition risk    -   For patients recovering from illness/surgery    -   For patients who need a low-residue diet-   Features:    -   Convenient, easy to mix    -   Low in saturated fat    -   Contains 9 g of total fat and <5 mg of cholesterol per serving    -   High in vitamins and minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested-   Ingredients: -D Corn Syrup, Maltodextrin (Corn), Sugar (Sucrose),    Corn Oil, Sodium and Calcium Caseinates, Soy Protein Isolate,    Artificial Flavor, Potassium Citrate, Magnesium Chloride, Sodium    Citrate, Calcium Phosphate Tribasic, Potassium Chloride, Soy    Lecithin, Ascorbic Acid, Choline Chloride, Zinc Sulfate, Ferrous    Sulfate, Alpha-Tocopheryl Acetate, Niacinamide, Calcium    Pantothenate, Manganese Sulfate, Thiamine Chloride Hydrochloride,    Cupric Sulfate, Pyridoxine Hydrochloride, Riboflavin, Vitamin A    Palmitate, Folic Acid, Biotin, Sodium Molybdate, Chromium Chloride,    Potassium Iodide, Sodium Selenate, Phylloquinone, Vitamin D3 and    Cyanocobalamin.-   Protein:    The protein source is a blend of two high-biologic-value proteins:    casein and soy.

Sodium and calcium caseinates 84% Soy protein isolate 16%

-   Fat:

The fat source is corn oil.

Corn oil 100%

-   Carbohydrate:

ENSURE POWDER contains a combination of corn syrup, maltodextrin, andsucrose. The mild sweetness of ENSURE POWDER, plus VARI-FLAVORS® FlavorPacs in pecan, cherry, strawberry, lemon, and orange, helps to preventflavor fatigue and aid in patient compliance.

-   Vanilla:

Corn Syrup 35% Maltodextrin 35% Sucrose 30%H. ENSURE® PUDDING

-   Usage: ENSURE PUDDING is a nutrient-dense supplement providing    balanced nutrition in a nonliquid form to be used with or between    meals. It is appropriate for consistency-modified diets (e.g., soft,    pureed, or full liquid) or for people with swallowing impairments.    ENSURE PUDDING is gluten-free.-   Patient Conditions:    -   For patients on consistency-modified diets (e.g., soft, pureed,        or full liquid)    -   For patients with swallowing impairments-   Features:    -   Rich and creamy, good taste    -   Good source of essential vitamins and minerals    -   Convenient-needs no refrigeration    -   Gluten-free-   Nutrient Profile per 5 oz: Calories 250, Protein 10.9%, Total Fat    34.9%, Carbohydrate 54.2%-   Ingredients:-   Vanilla: -D Nonfat Milk, Water, Sugar (Sucrose), Partially    Hydrogenated Soybean Oil, Modified Food Starch, Magnesium Sulfate,    Sodium Stearoyl Lactylate, Sodium Phosphate Dibasic, Artificial    Flavor, Ascorbic Acid, Zinc Sulfate, Ferrous Sulfate,    Alpha-Tocopheryl Acetate, Choline Chloride, Niacinamide, Manganese    Sulfate, Calcium Pantothenate, FD&C Yellow # 5, Potassium Citrate,    Cupric Sulfate, Vitamin A Palmitate, Thiamine Chloride    Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, FD&C Yellow #6,    Folic Acid, Biotin, Phylloquinone, Vitamin D3 and Cyanocobalamin.-   Protein:

The protein source is nonfat milk.

Nonfat milk 100%

-   Fat:

The fat source is hydrogenated soybean oil.

Hydrogenated soybean oil 100%

-   Carbohydrate:

ENSURE PUDDING contains a combination of sucrose and modified foodstarch. The mild sweetness and flavor variety (vanilla, chocolate,butterscotch, and tapioca) help prevent flavor fatigue. The productcontains 9.2 grams of lactose per serving.

-   Vanilla and Other Nonchocolate Flavors:

Sucrose 56% Lactose 27% Modified food starch 17%

-   Chocolate:

Sucrose 58% Lactose 26% Modified food starch 16%I. ENSURE® WITH FIBER:

-   Usage: ENSURE WITH FIBER is a fiber-containing, nutritionally    complete liquid food designed for people who can benefit from    increased dietary fiber and nutrients. ENSURE WITH FIBER is suitable    for people who do not require a low-residue diet. It can be fed    orally or by tube, and can be used as a nutritional supplement to a    regular diet or, in appropriate amounts, as a meal replacement.    ENSURE WITH FIBER is lactose- and gluten-free, and is suitable for    use in modified diets, including low-cholesterol diets.-   Patient Conditions:    -   For patients who can benefit from increased dietary fiber and        nutrients-   Features:    -   New advanced formula-low in saturated fat, higher in vitamins        and minerals    -   Contains 6 g of total fat and <5 mg of cholesterol per serving    -   Rich, creamy taste    -   Good source of fiber    -   Excellent source of essential vitamins and minerals    -   For low-cholesterol diets    -   Lactose- and gluten-free-   Ingredients:-   Vanilla: -D Water; Maltodextrin (Corn), Sugar (Sucrose), Sodium and    Calcium Caseinates, Oat Fiber, High-Oleic Safflower Oil, Canola Oil,    Soy Protein Isolate, Corn Oil, Soy Fiber, Calcium Phosphate    Tribasic, Magnesium Chloride, Potassium Citrate, Cellulose Gel, Soy    Lecithin, Potassium Phosphate Dibasic, Sodium Citrate, Natural and    Artificial Flavors, Choline Chloride, Magnesium Phosphate, Ascorbic    Acid, Cellulose Gum, Potassium Chloride, Carrageenan, Ferrous    Sulfate, Alpha-Tocopheryl Acetate, Zinc Sulfate, Niacinamide,    Manganese Sulfate, Calcium Pantothenate, Cupric Sulfate, Vitamin A    Palmitate, Thiamine Chloride Hydrochloride, Pyridoxine    Hydrochloride, Riboflavin, Folic Acid, Chromium Chloride, Biotin,    Sodium Molybdate, Potassium Iodide, Sodium Selenate, Phylloquinone,    Vitamin D3 and Cyanocobalamin.-   Protein:

The protein source is a blend of two high-biologic-value proteins-caseinand soy.

Sodium and calcium caseinates 80% Soy protein isolate 20%

-   Fat:

The fat source is a blend of three oils: high-oleic safflower, canola,and corn.

High-oleic safflower oil 40% Canola oil 40% Corn oil 20%The level of fat in ENSURE WITH FIBER meets American Heart Association(AHA) guidelines. The 6 grams of fat in ENSURE WITH FIBER represent 22%of the total calories, with 2.01% of the fat being from saturated fattyacids and 6.7% from polyunsaturated fatty acids. These values are withinthe AHA guidelines of ≦30% of total calories from fat, <10% of thecalories from saturated fatty acids, and ≦10% of total calories frompolyunsaturated fatty acids.

-   Carbohydrate:

ENSURE WITH FIBER contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla, chocolate, and butterpecan), plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry,lemon, and orange, help to prevent flavor fatigue and aid in patientcompliance.

-   Vanilla and Other Nonchocolate Flavors:

Maltodextrin 66% Sucrose 25% Oat Fiber 7% Soy Fiber 2%

-   Chocolate:

Maltodextrin 55% Sucrose 36% Oat Fiber 7% Soy Fiber 2%

-   Fiber:

The fiber blend used in ENSURE WITH FIBER consists of oat fiber and soypolysaccharide. This blend results in approximately 4 grams of totaldietary fiber per 8-fl. oz can. The ratio of insoluble to soluble fiberis 95:5.

The various nutritional supplements described above and known to othersof skill in the art can be substituted and/or supplemented with thePUFAs produced in accordance with the present invention.

J. Oxepa™ Nutritional Product

Oxepa is a low-carbohydrate, calorically dense, enteral nutritionalproduct designed for the dietary management of patients with or at riskfor ARDS. It has a unique combination of ingredients, including apatented oil blend containing eicosapentaenoic acid (EPA from fish oil),γ-linolenic acid (GLA from borage oil), and elevated antioxidant levels.

-   Caloric Distribution:    Caloric density is high at 1.5 Cal/mL (355 Cal/8 fl oz), to minimize    the volume required to meet energy needs. The distribution of    Calories in Oxepa is shown in Table A.

TABLE A Caloric Distribution of Oxepa per 8 fl oz. per liter % of CalCalories 355 1,500 — Fat (g) 22.2 93.7 55.2 Carbohydrate (g) 25 105.528.1 Protein (g) 14.8 62.5 16.7 Water (g) 186 785 —

-   Fat:    -   Oxepa contains 22.2 g of fat per 8-fl oz serving (93.7 g/L).    -   The fat source is an oil blend of 31.8% canola oil, 25%        medium-chain triglycerides (MCTs), 20% borage oil, 20% fish oil,        and 3.2% soy lecithin. The typical fatty acid profile of Oxepa        is shown in Table B.    -   Oxepa provides a balanced amount of polyunsaturated,        monounsaturated, and saturated fatty acids, as shown in Table        VI.    -   Medium-chain trigylcerides (MCTs)—25% of the fat blend—aid        gastric emptying because they are absorbed by the intestinal        tract without emulsification by bile acids.        The various fatty acid components of Oxepa™ nutritional product        can be substituted and/or supplemented with the PUFAs produced        in accordance with this invention.

TABLE B Typical Fatty Acid Profile Fatty Acids % Total g/8 fl oz* 9/L*Caproic (6:0) 0.2 0.04 0.18 Caprylic (8:0) 14.69 3.1 13.07 Capric (10:0)11.06 2.33 9.87 Palmitic (16:0) 5.59 1.18 4.98 Palmitoleic 1.82 0.381.62 Stearic 1.94 0.39 1.64 Oleic 24.44 5.16 21.75 Linoleic 16.28 3.4414.49 α-Linolenic 3.47 0.73 3.09 γ-Linolenic 4.82 1.02 4.29Eicosapentaenoic 5.11 1.08 4.55 n-3-Docosapent-aenoic 0.55 0.12 0.49Docosahexaenoic 2.27 0.48 2.02 Others 7.55 1.52 6.72 Fatty acids equalapproximately 95% of total fat.

TABLE C Fat Profile of Oxepa % of total calories from fat 55.2Polyunsaturated fatty acids 31.44 g/L Monounsaturated fatty acids 25.53g/L Saturated fatty acids 32.38 g/L n-6 to n-3 ratio 1.75:1 Cholesterol9.49 mg/8 fl oz 40.1 mg/L

-   Carbohydrate:    -   The carbohydrate content is 25.0 g per 8-fl-oz serving (105.5        g/L).    -   The carbohydrate sources are 45% maltodextrin (a complex        carbohydrate) and 55% sucrose (a simple sugar), both of which        are readily digested and absorbed.    -   The high-fat and low-carbohydrate content of Oxepa is designed        to minimize carbon dioxide (CO2) production. High CO2 levels can        complicate weaning in ventilator-dependent patients. The low        level of carbohydrate also may be useful for those patients who        have developed stress-induced hyperglycemia.    -   Oxepa is lactose-free.

Dietary carbohydrate, the amino acids from protein, and the glycerolmoiety of fats can be converted to glucose within the body. Throughoutthis process, the carbohydrate requirements of glucose-dependent tissues(such as the central nervous system and red blood cells) are met.However, a diet free of carbohydrates can lead to ketosis, excessivecatabolism of tissue protein, and loss of fluid and electrolytes. Theseeffects can be prevented by daily ingestion of 50 to 100 g of digestiblecarbohydrate, if caloric intake is adequate. The carbohydrate level inOxepa is also sufficient to minimize gluconeogenesis, if energy needsare being met.

-   Protein:    -   Oxepa contains 14.8 g of protein per 8-fl-oz serving (62.5 g/L).    -   The total calorie/nitrogen ratio (150:1) meets the need of        stressed patients.    -   Oxepa provides enough protein to promote anabolism and the        maintenance of lean body mass without precipitating respiratory        problems. High protein intakes are a concern in patients with        respiratory insufficiency. Although protein has little effect on        CO2 production, a high protein diet will increase ventilatory        drive.    -   The protein sources of Oxepa are 86.8% sodium caseinate and        13.2% calcium caseinate.    -   The amino acid profile of the protein system in Oxepa meets or        surpasses the standard for high quality protein set by the        National Academy of Sciences.    -   Oxepa is gluten-free.

1. An isolated nucleic acid sequence comprising or complementary to anucleotide sequence encoding a polypeptide having desaturase activity,wherein the amino acid sequence of said polypeptide comprises SEQ IDNO:26.
 2. An isolated nucleic acid sequence comprising or complementaryto a nucleotide sequence comprising SEQ ID NO:25.
 3. The isolatednucleic acid sequence of claim 2, wherein said sequence encodes afunctionally active desaturase which utilizes a polyunsaturated fattyacid as a substrate.
 4. The isolated nucleic acid sequence of claim 1,wherein said sequence is from Saprolenia diclina.